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copy 2015 Pearson Education Inc
Chapter 7
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The Structure and Replication of Genomes
bull Genetics bull Study of inheritance and inheritable traits as expressed
in an organisms genetic material
bull Genome bull The entire genetic complement of an organism
bull Includes its genes and nucleotide sequences
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Figure 71 The structure of nucleic acids
Hydrogen bond
SugarAdenine (A)nucleoside
Thymine (T)nucleoside
AndashT base pair (DNA) AndashU base pair (RNA)
Adenine (A)nucleoside
Uracil (U)nucleoside
Guanine (G)nucleoside
Cytosine (G)nucleoside
Double-stranded DNA
3prime end
5prime end3prime end
Adenine Thymine
5 endprime
Thymine nucleoside
Guanine Cytosine
GndashC base pair (DNA and RNA)
5prime end 5prime end3prime end
Thymine nucleotide
T A
CG
A T
G
3prime end
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Prokaryotic chromosomes
bull Main portion of DNA along with associated proteins and
RNA
bull Prokaryotic cells are haploid (single chromosome copy)
bull Typical chromosome is circular molecule of DNA in
nucleoid
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Figure 72 Bacterial genome
Nucleoid
Bacterium
Chromosome
Plasmid
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Plasmids
bull Small molecules of DNA that replicate independently
bull Not essential for normal metabolism growth or reproduction
bull Can confer survival advantages
bull Many types of plasmids
bull Fertility factors
bull Resistance factors
bull Bacteriocin factors
bull Virulence plasmids
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Nuclear chromosomes
bull Typically have more than one chromosome per cell
bull Chromosomes are linear and sequestered within nucleus
bull Eukaryotic cells are often diploid (two chromosome
copies)
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull Genetics bull Study of inheritance and inheritable traits as expressed
in an organisms genetic material
bull Genome bull The entire genetic complement of an organism
bull Includes its genes and nucleotide sequences
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Figure 71 The structure of nucleic acids
Hydrogen bond
SugarAdenine (A)nucleoside
Thymine (T)nucleoside
AndashT base pair (DNA) AndashU base pair (RNA)
Adenine (A)nucleoside
Uracil (U)nucleoside
Guanine (G)nucleoside
Cytosine (G)nucleoside
Double-stranded DNA
3prime end
5prime end3prime end
Adenine Thymine
5 endprime
Thymine nucleoside
Guanine Cytosine
GndashC base pair (DNA and RNA)
5prime end 5prime end3prime end
Thymine nucleotide
T A
CG
A T
G
3prime end
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Prokaryotic chromosomes
bull Main portion of DNA along with associated proteins and
RNA
bull Prokaryotic cells are haploid (single chromosome copy)
bull Typical chromosome is circular molecule of DNA in
nucleoid
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Figure 72 Bacterial genome
Nucleoid
Bacterium
Chromosome
Plasmid
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Plasmids
bull Small molecules of DNA that replicate independently
bull Not essential for normal metabolism growth or reproduction
bull Can confer survival advantages
bull Many types of plasmids
bull Fertility factors
bull Resistance factors
bull Bacteriocin factors
bull Virulence plasmids
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Nuclear chromosomes
bull Typically have more than one chromosome per cell
bull Chromosomes are linear and sequestered within nucleus
bull Eukaryotic cells are often diploid (two chromosome
copies)
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 71 The structure of nucleic acids
Hydrogen bond
SugarAdenine (A)nucleoside
Thymine (T)nucleoside
AndashT base pair (DNA) AndashU base pair (RNA)
Adenine (A)nucleoside
Uracil (U)nucleoside
Guanine (G)nucleoside
Cytosine (G)nucleoside
Double-stranded DNA
3prime end
5prime end3prime end
Adenine Thymine
5 endprime
Thymine nucleoside
Guanine Cytosine
GndashC base pair (DNA and RNA)
5prime end 5prime end3prime end
Thymine nucleotide
T A
CG
A T
G
3prime end
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Prokaryotic chromosomes
bull Main portion of DNA along with associated proteins and
RNA
bull Prokaryotic cells are haploid (single chromosome copy)
bull Typical chromosome is circular molecule of DNA in
nucleoid
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Figure 72 Bacterial genome
Nucleoid
Bacterium
Chromosome
Plasmid
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Plasmids
bull Small molecules of DNA that replicate independently
bull Not essential for normal metabolism growth or reproduction
bull Can confer survival advantages
bull Many types of plasmids
bull Fertility factors
bull Resistance factors
bull Bacteriocin factors
bull Virulence plasmids
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Nuclear chromosomes
bull Typically have more than one chromosome per cell
bull Chromosomes are linear and sequestered within nucleus
bull Eukaryotic cells are often diploid (two chromosome
copies)
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Prokaryotic chromosomes
bull Main portion of DNA along with associated proteins and
RNA
bull Prokaryotic cells are haploid (single chromosome copy)
bull Typical chromosome is circular molecule of DNA in
nucleoid
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Figure 72 Bacterial genome
Nucleoid
Bacterium
Chromosome
Plasmid
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Plasmids
bull Small molecules of DNA that replicate independently
bull Not essential for normal metabolism growth or reproduction
bull Can confer survival advantages
bull Many types of plasmids
bull Fertility factors
bull Resistance factors
bull Bacteriocin factors
bull Virulence plasmids
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Nuclear chromosomes
bull Typically have more than one chromosome per cell
bull Chromosomes are linear and sequestered within nucleus
bull Eukaryotic cells are often diploid (two chromosome
copies)
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 72 Bacterial genome
Nucleoid
Bacterium
Chromosome
Plasmid
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Plasmids
bull Small molecules of DNA that replicate independently
bull Not essential for normal metabolism growth or reproduction
bull Can confer survival advantages
bull Many types of plasmids
bull Fertility factors
bull Resistance factors
bull Bacteriocin factors
bull Virulence plasmids
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Nuclear chromosomes
bull Typically have more than one chromosome per cell
bull Chromosomes are linear and sequestered within nucleus
bull Eukaryotic cells are often diploid (two chromosome
copies)
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull The Structure of Prokaryotic Genomesbull Plasmids
bull Small molecules of DNA that replicate independently
bull Not essential for normal metabolism growth or reproduction
bull Can confer survival advantages
bull Many types of plasmids
bull Fertility factors
bull Resistance factors
bull Bacteriocin factors
bull Virulence plasmids
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Nuclear chromosomes
bull Typically have more than one chromosome per cell
bull Chromosomes are linear and sequestered within nucleus
bull Eukaryotic cells are often diploid (two chromosome
copies)
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Nuclear chromosomes
bull Typically have more than one chromosome per cell
bull Chromosomes are linear and sequestered within nucleus
bull Eukaryotic cells are often diploid (two chromosome
copies)
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 73 Eukaryotic nuclear chromosomal packaging
10 nm
HistonesLinkerDNA
DNA
10 nm
Nucleosomes Chromatin fiber Euchromatin andheterochromatin
Highly condensedduplicated chromosome of dividing nucleus
Active(loosely packed)
Inactive(tightlypacked)
Nucleosome
30 nm 700 nm1400 nm
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull The Structure of Eukaryotic Genomesbull Extranuclear DNA of eukaryotes
bull DNA molecules of mitochondria and chloroplasts
bull Resemble chromosomes of prokaryotes
bull Code only for about 5 of RNA and proteins
bull Some fungi algae and protozoa carry plasmids
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull DNA Replicationbull Key to replication is complementary structure of the two
strands
bull Replication is semiconservative
bull New DNA composed of one original and one daughter
strand
bull Anabolic polymerization process that requires
monomers and energy
bull Triphosphate deoxyribonucleotides serve both functions
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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DNA Replication Overview
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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DNA Replication
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 74 Semiconservative model of DNA replication
OriginalDNA
Firstreplication
Secondreplication
Original strand
New strands
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Guanosine triphosphate deoxyribonucleotide (dGTP)
Guanine nucleotide (dGMP)
High-energybond
DeoxyriboseGuanine base
Guanosine (nucleoside)
Existing DNA strand Triphosphatenucleotide
Diphosphate releasedenergy used for synthesis
Longer DNA strand
OH
Figure 75 The dual role of triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull DNA Replicationbull Initial processes in bacterial DNA replication
bull Replication begins at the origin
bull DNA polymerase replicates DNA only 5prime to 3prime
bull Because strands are antiparallel new strands are
synthesized differently
bull Leading strand synthesized continuously
bull Lagging strand synthesized discontinuously
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 76a DNA replication
Chromosomal proteins(histones in eukaryotes andarchaea) removed
DNA helicase
Replication fork
DNA polymerase III
Initial processesStabilizing proteins
3prime
5prime
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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DNA Replication Replication Proteins
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Primase
RNA primer
Leading strand
Triphosphatenucleotide
Replication fork
Synthesis of leading strand
Replication fork
Triphosphatenucleotide
Okazakifragment Lagging
strand
DNA ligaseDNA polymerase IDNA polymerase IIIPrimase
RNAprimer
Synthesis of lagging strand
98
76
10
2
3 1
P P+
3prime5prime
3prime5prime
Figure 76b-c DNA replication
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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DNA Replication Forming the Replication Fork
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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DNA Replication Synthesis
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull DNA Replicationbull Other characteristics of bacterial DNA replication
bull Bidirectional
bull Gyrases and topoisomerases remove supercoils in DNA
bull DNA is methylated
bull Control of genetic expression
bull Initiation of DNA replication
bull Protection against viral infection
bull Repair of DNA
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 77 The bidirectionality of DNA replication in prokaryotes
Origin Parentalstrand
Daughterstrand
Replication forks
Replicationproceeds in both directions Termination
of replication
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull DNA Replicationbull Replication of eukaryotic DNA
bull Similar to bacterial replication
bull Some differences
bull Uses four DNA polymerases
bull Thousands of replication origins
bull Shorter Okazaki fragments
bull Plant and animal cells methylate only cytosine bases
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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The Structure and Replication of Genomes
bull Tell Me Whybull DNA replication requires a large amount of energy yet
none of a cells ATP energy supply is used Why isnt it
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull The Relationship Between Genotype and
Phenotypebull Genotype
bull Set of genes in the genome
bull Phenotype
bull Physical features and functional traits of the organism
bull Genotype determines phenotype
bull Not all genes are active at all times
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull The Transfer of Genetic Informationbull Transcription
bull Information in DNA is copied as RNA
bull Translation
bull Polypeptides are synthesized from RNA
bull Central dogma of genetics
bull DNA is transcribed to RNA
bull RNA is translated to form polypeptides
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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copy 2015 Pearson Education Inc
Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 78 The central dogma of genetics
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Transcription Overview
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Translation Overview
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull The Events in Transcriptionbull Five types of RNA transcribed from DNA
bull RNA primersbull mRNAbull rRNAbull tRNAbull Regulatory RNA
bull Occur in nucleoid of prokaryotesbull Three steps
bull Initiation bull Elongation bull Termination
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 79a The events in the transcription of RNA in prokaryotes
RNA polymerase attachesnonspecifically to DNA andtravels down its length untilit recognizes a promotersequence Sigma factorenhances promoterrecognition in bacteria
Upon recognition of thepromoter RNA polymeraseunzips the DNA moleculebeginning at the promoter
Unzipping of DNA movement of RNA polymerase
Attachment of RNA polymerase
Sigma factorPromoter
RNA polymerase
Bubble
Terminator
TemplateDNA strand
DNA
Initiation of transcription
5prime
3prime
5prime
5prime 3prime
3prime5prime
3prime
1a
1b
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 79b The events in the transcription of RNA in prokaryotes
Triphosphate ribonucleotidesalign with their DNAcomplements and RNApolymerase links themtogether synthesizing RNANo primer is needed Thetriphosphate ribonucleotidesalso provide the energyrequired for RNA synthesis
Elongation of the RNA transcript
Growing RNA molecule(transcript)
Bubble
TemplateDNAstrand
2
5prime
3prime
3prime
5prime5prime
5prime3prime
3prime
3prime
5prime
PP P
PPP
CG A
T A C C A C CAG
GUGGU
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 710 Concurrent RNA transcription
3prime 3prime 3prime 3prime 3prime 3prime 3prime 3prime5prime
5prime
5prime5prime5prime
5prime
5prime
5prime
Promoter
RNA polymerases
Sigma factor
RNA
Template DNAstrand
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 79c The events in the transcription of RNA in prokaryotes
3prime5prime
5prime
3prime
3prime
3prime
5prime
Self-termination transcription of GC-rich terminatorregion produces a hairpin loop which creates tensionloosening the grip of the polymeraseon the DNA
Rho-dependant termination Rho pushes between polymeraseand DNA This causes release of polymerase RNA transcriptand Rho
GC-richhairpinloop
Termination of transcription release of RNA polymerase
Templatestrand
Rho protein movesalong RNA
Rho terminationprotein
RNA polymerase
RNA transcriptreleased
Terminator
CC CCCG GAAAAAAAAT
UUUUUUUUU
3b3a
TerminatorTerminator
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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copy 2015 Pearson Education Inc
Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Transcription The Process
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull The Events in Transcriptionbull Transcriptional differences in eukaryotes
bull RNA transcription occurs in the nucleus
bull Transcription also occurs in mitochondria and chloroplasts
bull Three types of nuclear RNA polymerases
bull Numerous transcription factors
bull mRNA is processed before translation
bull Capping
bull Polyadenylation
bull Splicing
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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3prime
3prime
3prime 5prime
5prime
5prime
5prime
Introns (noncoding regions)
TemplateDNA strand
Exon 1
cap Intron 1 Intron 2 Intron 3
Exon 2 Exon 3Pre-mRNA
Poly-A tail
Exon 1
Spliceosomes
Exons (polypeptide coding regions)
Processing
mRNA splicing
mRNA (codes forone polypeptide)
Nuclear envelopeNucleoplasm
Cytosol
mRNA
Nuclear pore
Exon 2Exon 3
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
AAAAAAAAAAAAAA
A
Intron 1
AAAAA
AAAAAA AA
Transcription
Figure 711 Processing eukaryotic mRNA
AA A AA
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull Translationbull Process in which ribosomes use genetic information of
nucleotide sequences to synthesize polypeptides
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 712 The genetic code
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Translation Genetic Code
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Protein Synthesis
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull Translationbull Participants in translation
bull Messenger RNA
bull Transfer RNA
bull Ribosomes and ribosomal RNA
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 713 A single prokaryotic mRNA can code for several polypeptides
5prime
5prime3prime
3prime
Promoter Gene 1 Gene 2 Gene 3 TerminatorTemplateDNA strand
StartcodonAUG
StartcodonAUG
StartcodonAUGUAA UAG UAA
Ribosomebindingsite (RBS)
Stopcodon
RBS Stopcodon
RBS Stopcodon
UntranslatedmRNA
mRNA
Polypeptide 1 Polypeptide 2 Polypeptide 3
Translation
Transcription
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 714 Transfer RNA
Acceptorstem
Hydrogenbonds
Hairpinloops
tRNA icon
Anticodon
Anticodon
5prime5prime
3prime
3prime
A C C
OH
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 715 Ribosomal structures
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 716 Assembled ribosome and its tRNA-binding sites
Largesubunit
mRNA
Smallsubunit
Prokaryotic ribosome(angled view) attachedto mRNA
Largesubunit
Nucleotidebases
Prokaryotic ribosome(schematic view) showingtRNA-binding sites
Smallsubunit
tRNA-bindingsites
Esite
Psite
Asite
mRNA
5prime 3prime
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull Translationbull Events in translation
bull Three stages of translation
bull Initiation
bull Elongation
bull Termination
bull All stages require additional protein factors
bull Initiation and elongation require energy (GTP)
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Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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copy 2015 Pearson Education Inc
Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 717 The initiation of translation in prokaryotes
fMetInitiatortRNA
AnticodonmRNA Start codon
Smallribosomalsubunit
GTP GDP P
fMet
tRNA
fMet
Initiation complex
5prime 3prime
fMet
Largeribosomalsubunit
UU U U AU GGA CA C
AP
U U U AU GGA C
AP
U U U AU GGA CU A C
AP
U AC
1 2 3
E
+
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Protein Synthesis
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
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Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 718 The elongation stage of translation
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Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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copy 2015 Pearson Education Inc
Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
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Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 719 In prokaryotes a polyribosomemdashone mRNA and many ribosomes and polypeptides
mRNA Ribosomes Polypeptides mRNA Ribosomes Polypeptides
Direction oftranscription
copy 2015 Pearson Education Inc
Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
copy 2015 Pearson Education Inc
Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Protein Synthesis
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
copy 2015 Pearson Education Inc
Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull Translationbull Events in translation
bull Termination
bull Release factors recognize stop codons
bull Modify ribosome to activate ribozymes
bull Ribosome dissociates into subunits
bull Polypeptides released at termination may function
alone or together
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Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Translation The Process
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Protein Synthesis
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
copy 2015 Pearson Education Inc
Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
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Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull Translationbull Translation differences in eukaryotes
bull Initiation occurs when ribosomal subunit binds to 5prime
guanine cap
bull First amino acid is methionine rather than f-methionine
bull Ribosomes can synthesize polypeptides into the cavity of
the rough endoplasmic reticulum
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copy 2015 Pearson Education Inc
Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
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Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
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copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
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Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
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Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Protein Synthesis
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
copy 2015 Pearson Education Inc
Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
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Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Protein Synthesis
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Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
copy 2015 Pearson Education Inc
Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Most genes are expressed at all times
bull Other genes are transcribed and translated when cells
need them
bull Allows cell to conserve energy
bull Quorum sensing regulates production of some proteins
bull Detection of secreted quorum-sensing molecules can
signal bacteria to synthesize a certain protein
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
copy 2015 Pearson Education Inc
Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
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Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
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Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Regulation of polypeptide synthesis
bull Typically halts transcription
bull Can stop translation directly
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
copy 2015 Pearson Education Inc
Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
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Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull An operon consists of a promoter and a series of genes
bull Controlled by a regulatory element called an operator
bull Typically polycistronic (code for several polypeptides)
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
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Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
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Operons Overview
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Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
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Operons Repression
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
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Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
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Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
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Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
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Mutations Types
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Figure 724 The effects of the various types of point mutations
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Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
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Mutagens
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Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
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Figure 726 The structure and effects of a nucleotide analog
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Figure 727 The action of a frameshift mutagen
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Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
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Mutations Repair
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Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
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Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
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Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
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Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
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Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
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Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
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Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
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Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
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Horizontal Gene Transfer Overview
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
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Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
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Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
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Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
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Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
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Figure 720 An operon
OperonPromoter Operator Structural genes
Template DNA strandRegulatory gene
5prime43213prime
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
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copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
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Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
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Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull Nature of prokaryotic operons
bull Inducible operons must be activated by inducers
bull Lactose operon
bull Repressible operons are transcribed continually until
deactivated by repressors
bull Tryptophan operon
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
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Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Operons Overview
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Template DNAstrand
Template DNA strand
RNA polymerase
lac operon
Operator(blocked)PromoterPromoter and
regulatory gene
Continual transcription
Repressor mRNA
Repressor
lac operon repressed
Lactose catabolism genes
TranscriptionproceedsRepressor
cannot bind
Repressor
Inactivatedrepressor
Inducer (allolactosefrom lactose)
lac operon induced
mRNA forlactose catabolism
2 31
3prime
3prime 5prime
5prime
5prime
RNApolymerasecannotbind
1
2
3
4
2 31
Continual translation
Repressor mRNA
Figure 721 The lac operon an inducible operon
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Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
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Operons Induction
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Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 722 CAP-cAMP enhances lac transcription
CAP binding site
lac genes
RNA polymerase
Transcription proceeds
Promoter Operator
cAMP bound to CAP
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Operons Induction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 723 The trp operon a repressible operon
Regulatory gene
mRNA
Inactive repressor
trp operon active
Promoter
trp operon with five genes
Transcription
Enzymes of tryptophan biosynthetic pathway
Template DNA strand
Tryptophan
Operator blocked
Inactiverepressor
trp operon repressed
Trp
3prime
Movement of RNA polymerase ceases
Trp
Tryptophan (corepressor) Activated
repressor
mRNA coding multiple polypeptides5prime 3prime
Operator
5prime
1 2 3 4 55prime
3prime
1 2 3 4 55prime
TrpTrp
Trp
Trp
3prime
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Operons Repression
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
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Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
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Transduction Generalized Transduction
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Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
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Conjugation Overview
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Conjugation F Factor
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F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
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Conjugation Hfr Conjugation
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Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
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Transposons Overview
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Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
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Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
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Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
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Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull microRNAs
bull Produced by eukaryotic cells
bull Bind regulatory proteins to form miRNA-induced
silencing complex (miRISC)
bull Bind complementary mRNA and inhibit its
translation
bull Regulates several cellular processes
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
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Transposons Complex Transposons
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Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Gene Function
bull Regulation of Genetic Expressionbull RNA molecules can control translation
bull Regulatory RNAs can regulate translation of polypeptides
bull Short interference RNA (siRNA)
bull RNA molecule complementary to a portion of mRNA tRNA or DNA
bull Binds RISC proteins to form siRISC
bull siRISC binds and cuts the target nucleic acid
bull Riboswitch
bull RNA molecule that changes shape to help regulate translation
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Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Gene Function
bull Tell Me Whybull In bacteria polypeptide translation can begin even
before mRNA transcription is complete Why cant this
happen in eukaryotes
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutation bull Change in the nucleotide base sequence of a genome
bull Rare event
bull Almost always deleterious
bull Rarely leads to a protein that improves ability of
organism to survive
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations of Genes
bull Types of Mutationsbull Point mutations
bull One base pair is affected
bull Substitutions and frameshift mutations
bull Frameshift mutations
bull Nucleotide triplets after the mutation are displaced
bull Creates new sequence of codons
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations Types
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 724 The effects of the various types of point mutations
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations of Genes
bull Mutagensbull Radiation
bull Ionizing radiationbull Nonionizing radiation
bull Chemical mutagensbull Nucleotide analogs
bull Disrupt DNA and RNA replicationbull Nucleotide-altering chemicals
bull Alter the structure of nucleotidesbull Result in base-pair substitutions and missense
mutationsbull Frameshift mutagens
bull Result in nonsense mutations
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutagens
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 725 A pyrimidine (in this case thymine) dimer
Ultraviolet light
Thymine dimer
G
C
C T G T AGG
G A C A A C C A T
T T=
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 726 The structure and effects of a nucleotide analog
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 727 The action of a frameshift mutagen
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations of Genes
bull Frequency of Mutationbull Mutations are rare events
bull Otherwise organisms could not effectively reproduce
bull About 1 of every 10 million genes contains an error
bull Mutagens increase the mutation rate by a factor of 10 to
1000 times
bull Many mutations stop transcription or code for
nonfunctional proteins
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations Repair
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 728a-b DNA repair mechanisms
Visible light
Thymine dimer
Light-activated repair enzyme
Cut
Repairenzyme
Light repair
Dark repair
DNA polymerase Iand ligase repairthe gap
G A C A
C T G A A T
T
C T G A A T
G A C AT TT=
C T G A A T
G A C AT T
G
C
C T G A A T
GA C A
T T
G
C
C T G A A T
G A C AT T
G
C=
=
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 728c-d DNA repair mechanisms
Base excision repair enzymes remove incorrect nucleotide
DNA polymerase I and ligase repair gap
Mutated DNA(incorrect nucleotide pair)
Mismatch repair enzyme removes incorrect segment
DNA polymerase III correctly repairs the gap
Mismatch repair
Base-excision repair
C C G A A T
G G C AT T
A
T C G T
G C AC C G A A T
G G C AT T
A
C G T
G C AC C G A A T
G G C AT T
A
G C G T
G C A
G G A T
C AT T
C
G C G T
G C A G G A T
CAT T
C
G C G T
G C A G G A T
C AC T
C
G C G T
G C A
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations of Genes
bull Identifying Mutants Mutagens and Carcinogens
bull Mutants
bull Descendants of a cell that does not repair a mutation
bull Wild types
bull Cells normally found in nature
bull Methods to recognize mutants
bull Positive selection
bull Negative (indirect) selection
bull Ames test
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 729 Positive selection of mutants
Medium with penicillin (only penicillin-resistant cell grows into colony)
Medium without penicillin (both types of cells form colonies)
Penicillin-sensitive cells
Penicillin- resistant cell
Penicillin- resistantmutants indistinguishable from nonmutants
Medium with penicillin Medium withoutpenicillin
Mutagen induces mutations
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 730 The use of negative (indirect) selection to isolate a tryptophan auxotroph
Bacteria
Stamp replica plates with velvet
Complete medium containing tryptophan
Medium lacking tryptophan
IncubationIdentify auxotrophas colony growing on complete medium but not on lacking medium
Tryptophan auxotroph cannot grow
All colonies grow
Inoculate auxotroph colony into complete medium
X
X X
XX
4
5
6
Bacterial suspension
Bacterial colonies grow A few may be tryptophan auxotrophs Most are wild type
Incubation
Mutagen
Inoculate bacteria onto complete medium containing tryptophan
Stamp sterile velvet onto plate picking up cells from each colony
Sterile velvet surface
3
2
1
X
X
X
X
X
X
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 731 The Ames test
Colony of revertant (his+) Salmonella
No growth
Incubation
Medium lacking
histidine
Liver extract
Experimental tube
Suspected mutagen
Liver extract
Control tube
Culture of hisndash Salmonella
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Mutations of Genes
bull Tell Me Whybull Changes in RNA resulting from poor transcription of
RNA to DNA are not as deleterious to an organism as
changes to its DNA resulting from mutations Why is this
the case
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Exchange of nucleotide sequences often occurs
between homologous sequences
bull Recombinants
bull Cells with DNA molecules that contain new nucleotide
sequences
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 732 Genetic recombination
Homologous sequences
Enzyme nicks one strand of DNA at homologous sequence
Recombination enzyme inserts the cut strand into second molecule which is nicked in the process
Ligase anneals nicked ends in new combinations
Molecules resolve into recombinants
Recombinant A
Recombinant B
3prime DNA A
DNA B5prime3prime5prime
A
B
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotes
bull Vertical gene transfer
bull Passing of genes to the next generation
bull Horizontal gene transfer
bull Donor cell contributes part of genome to recipient cell
bull Three types
bull Transformation
bull Transduction
bull Bacterial conjugation
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Horizontal Gene Transfer Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transformation
bull Recipient cell takes up DNA from the environment
bull Provided evidence that DNA is genetic material
bull Cells that take up DNA are competent
bull Results from alterations in cell wall and cytoplasmic
membrane that allow DNA to enter cell
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 733 Transformation of Streptococcus pneumoniae
Observations of Streptococcus pneumoniae
Live cellsInjection
Capsule
Heat-treateddead cells ofstrain S Injection
Strain R live cells(no capsule)
Injection
Griffiths experimentLivingstrain R
Mouse dies
Mouse lives
Mouse lives
Heat-treateddead cellsof strain S
Injection
Mouse dies
Culture ofStreptococcusfrom deadmouse
Living cellswith capsule(strain S)
In vitro transformation
Heat-treateddead cells ofstrain S
DNA brokeninto pieces
DNA fragmentfrom strain S
Living strain R
Some cells takeup DNA from theenvironment andincorporate it intotheir chromosomes
Transformed cellsacquire ability tosynthesize capsules
+
XXXX
XXXX XXXX
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Transformation
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Transfer of DNA from one cell to another via replicating
virus
bull Virus must be able to infect both donor and recipient cells
bull Virus that infects bacteria called a bacteriophage (phage)
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 734 Transduction
Phage injects its DNA
Phage enzymesdegrade host DNA
PhageDNA
Cell synthesizes newphages that incorporatephage DNA and mistakenlysome host DNA
Transducing phageinjects donor DNA
Donor DNA is incorporatedinto recipients chromosomeby recombination
InsertedDNA
Transduced cell
Recipient host cell
Transducing phage
Phage with donor DNA(transducing phage)
Bacterial chromosome
BacteriophageHost bacterial cell(donor cell)
1
2
3
4
5
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Transduction
bull Generalized transduction
bull Transducing phage carries random DNA segment
from donor to recipient
bull Specialized transduction
bull Only certain donor DNA sequences are transferred
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Transduction Generalized Transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Transduction Specialized Transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Horizontal Gene Transfer Among Prokaryotesbull Conjugation
bull Genetic transfer requires physical contact between the
donor and recipient cell
bull Donor cell remains alive
bull Mediated by conjugation (sex) pili
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Conjugation Overview
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Conjugation F Factor
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
F plasmid Origin oftransfer
Pilus Chromosome
F
Donor cell attaches to a recipient cell withits pilus
Pilus may draw cells together
One strand of F plasmid DNA transfersto the recipient
The recipient synthesizes a complementarystrand to become an F+ cell with a pilus thedonor synthesizes a complementary strandrestoring its complete plasmid
Pilus
1
+ cell
2
F+ cell
3
4
F_
cellF+ cell
Figure 735 Bacterial conjugation
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 736 Conjugation involving an Hfr cellDonor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid Donor DNA Part of F plasmid
F recipient
Incomplete F plasmidcell remains Fminus
F plasmid integratesinto chromosome byrecombination
Cells join via a pilus
Portion of F plasmid partiallymoves into recipient celltrailing a strand of donorsDNA
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell cells synthesizecomplementary DNA strands
Donor DNA and recipientDNA recombine making arecombinant F cell
Recombinant cell (still Fminus )
ndash
1
2
3
4
5ndash
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Conjugation Hfr Conjugation
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Conjugation Chromosome Mapping
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Transposons
bull Segments of DNA that move from one location to another
in the same or different molecule
bull Result is a kind of frameshift insertion (transpositions)
bull Transposons all contain palindromic sequences at each
end
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 737 Transposition
DNATransposon
Jumping transposons Transposons move from one place to another on a DNA molecule
Replicating transposons Transposons may replicate while moving resulting in more transposons in the cell
Transposons can move onto plasmidsTransposons moving onto plasmids can be transferred to another cell
Plasmid withtransposon
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Transposons Overview
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Transposons and Transpositionbull Simplest transposons
bull Insertion sequences
bull Have no more than two inverted repeats and a gene for
transposase
bull Complex transposons
bull Contain one or more genes not connected with
transposition
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Figure 738 TransposonsTransposon Insertion sequence IS1
Inverted repeat (IR) Transposase gene Inverted repeat (IR)
Target site
DNAmolecule
IS1
Target IS1
Transposase
Target site Copy of IS1
Copy oftarget site
OriginalIS1
Complex transposon
IS1
Kanamycin-resistancegene IS1
IR
A C T GT A C T TAT G A A T G A C T A
TA A
A AAATT
TTTTAC G G
G C C
IRIRIR
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Transposons Insertion Sequences
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Transposons Complex Transposons
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Genetic Recombination and Transfer
bull Tell Me Whybull Why is the genetic ancestry of microbes much more
difficult to ascertain than the ancestry of animals
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction
copy 2015 Pearson Education Inc
Important topics
bull Structure of chromosomendash DNAndash Histonndash Chromatidndash Nucleosome
bull Comparison between bacterial and eukaryotic chromosomebull Bacterial plasmid
ndash Structure ndash Function
bull Replication processbull Transcription processbull Translation processbull Different forms of mutation
ndash Frameshiftndash Silentndash Missensendash Nonsense
bull Transformation vs conjugation and transduction