IN THE NAME OF IN THE NAME OF GODGOD

Microbial Genetic

After completion sessions we should be able to describe:

• Bacteria gene structure• Replication in bacteria• Bacterial DNA structure• Transfer of DNAin bacteria• Bacterial recombinant• Bacterial mutation• Bacterial gene expression• Regulation mechanisms in Bacteria• Gel electrophoresis techniques• Hybridization• Genetic engineering

Dr. Nasr Esfahani

1940sDNA is genetic material

DNA: The Instruction Manual for all Life

Dr. Nasr Isfahani

gene, a segment of DNA, information for a specific biochemical or physiologic property:

eye color ,resistance to an antibiotic,

variation in phenotype: change in genotype,

selection of the gene requires its expression

Dr. Nasr Isfahani

Organization of Genes

Genetic information: (DNA, some RNA: viruses [RNA].

Most DNA :double-stranded, (A-T; G-C)

The orientation: antiparallel; one 5' to 3‘ its complementary strand runs 3' to 5'.

Each helical turn of the helix has one major groove and one minor groove.

Dr. Nasr Isfahani

Watson-Crick : helical sugar-phosphate backbones

the two strands held together by hydrogen bonding between the bases.

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Genetic Information: DNA: 5 kbp (virus), E.coli:4640

kbp, about 1 mm: bp –bp: 0.34 nm: super coiling

RNA: Some Bacteriophage and viruses: uracil/thymine

tRNA: <100 base, mRNA: >thousands baserRNA: 120 bp(5S), 1540 bp(16S),

2900 bp(23S).tRNA and rRNA: 95%

Dr. Nasr Isfahani

a ribosome: one copy each of the 16S, 23S, and 5S RNAs as well as many proteins.

Dr. Nasr Isfahani

Each base pair is separated from the next by about 0.34 nm, or 3.4 x 10–7 mm, so that the total length of the E coli chromosome is roughly 1 mm.

dimensions of the bacterial cell are roughly 1000-fold smaller than this length: supercoiling,

Dr. Nasr Isfahani

DNA منفی بار توسط به حدودی های تا آمین پلی

منیزیوم یونهای و میشوند کوچک خنثیهیستون اما شبه های پروتئین

های هیستون با مشابه عملکردیعهده بر را اوکاریوتی کروماتین

میگیرند. وجود میتوزی دستگاه و هسته غشای

ندارد. تقریبی باز DNAدالتون 3 109وزن

باشد شده هاپلوئید واحد کروموزوم یک شکل در تقریبی طول .1با بود خواهد متر میلی

Dr. Nasr Esfahani

های کپی تعداد سلول، چرخه یک درچرخه مرحله به کروموزوم این . های کپی اگر دارد بستگی سلولخواهند یکسان باشند موجود متعددی

بود. ( قبیل از ها پروکاریوت از بعضی

بیماری عامل بورگدورفری، بورلیا . دارند خطی کروموزوم الیم

از بعضی در خطی کروموزومهاییافت نیز استرپتومیسس های گونه

. است شدهDr. Nasr Esfahani

DNA با خود انتهای یک در سلول

غشاء از بخشی فرورفتگیعنوان به که مزوزوم سیتوپالسم

. دارد ارتباط میشود شناخته

Dr. Nasr Esfahani

Prokaryotic Genome Prokaryotic Genome

Replicon:1- Circular 2 stranded DNA (580 kbp: Mycoplasma genitalium to more 4600 kbp: E. coli, MTB: 4410):

2- Plasmid: a few to 100 Kbp: for specific function

Haploid

Dr. Nasr Isfahani

3. Transposons: genetic elements, several kbp of DNA, including the information necessary for their migration from one genetic locus to another.

In doing so, they create insertion mutations.

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Dr. Nasr Isfahani

relatively short transposons (750–2000 bp long),

produces the majority of insertion mutations.

Dr. Nasr Isfahani

insertion sequenceinsertion sequence

The insertion elements (also known as [IS] elements) carry only the genes for enzymes needed to promote their own transposition.

Almost all bacteria carry IS elements, with each species harboring its own characteristic ones.

Dr. Nasr Isfahani

Related IS elements can sometimes be found in different bacteria.

Plasmids also carry IS elements, which are important in the formation of high-frequency recombinant (Hfr) strains.

Dr. Nasr Isfahani

genes for specialized functions : antibiotic resistance

Unlike plasmids, transposons do not contain genetic information necessary for their own replication.

Dr. Nasr Isfahani

Viral Genome Viral Genome

DNA: Two strands: Adenovirus Single strand : Parvovirus

Dr. Nasr Isfahani

RNA: Two strands : Reovirus Single strand :retrovirus: reverse

transcriptase Abnormal bases: Hydroxymethylcytosine

Lytic(T2, T4) or Lysogenic(temperate phages, prophage: lambda) Phase

Filamentous phages: (M13, E.coli) ssDNA

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Diagrams of phage T2 based on electron micrographic observation.

Dr. Nasr Isfahani

Dr. Nasr Isfahani

lytic and lysogenic growth

Replication in Bacteria

Dr. Nasr Isfahani

(a( Hypothesis 1:

Semi-conservative replication

(b( Hypothesis 2:Conservative replication

Intermediate molecule

(c( Hypothesis 3:Dispersive replication

Dr. Nasr Isfahani

Meselson and Stahl Semi-conservative replication of DNA

Isotopes of nitrogen (non-radioactive) were used in this experiment

Dr. Nasr Isfahani

Features of DNA ReplicationFeatures of DNA Replication

semiconservative◦Each strand of template DNA is being copied.

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Origin

5’3’

3’5’

UNIDIRECTIONAL REPLICATION

Origin

5’3’

3’5’

BIDIRECTIONAL REPLICATION

Replication can be Uni- or Bidirectional

(DNA: in bacteria: ori locus and (DNA: in bacteria: ori locus and ter locus)ter locus)

Two directionalBy replication forkIn plasmid : some times

unidirectional

Dr. Nasr Isfahani

Features of DNA ReplicationFeatures of DNA Replicationsemiconservative

◦Each strand of template DNA is being copied.

bidirectional◦Bidirectional replication involves two replication forks, which move in opposite directions

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Continuous synthesis

Discontinuous synthesis

DNA replication is semi-discontinuous

Features of DNA ReplicationFeatures of DNA Replicationsemiconservative

◦Each strand of template DNA is being copied.bidirectional

◦Bidirectional replication involves two replication forks, which move in opposite directions

semidiscontinuous◦The leading strand copies continuously◦The lagging strand copies in segments

(Okazaki fragments) which must be joined

Dr. Nasr Isfahani

Note:Note: DNAP I: functions in repair and replication

DNAP II: functions in DNA repair (proven in 1999)

DNAP III: principal DNA replication enzyme

DNAP IV: functions in DNA repair (discovered in 1999)

DNAP V: functions in DNA repair (discovered in 1999)

Dr. Nasr Isfahani

DNA Polymerase III DNA Polymerase III

The "real" replicative polymerase in E. coli

It’s fast: up to 1,000 dNTPs added/ sec/ enzyme

makes 1 error in 107 dNTPs added, proofreading, this gives a final error rate of 1 in 1010 overall.

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Gene Transmission

Gene Transmission

Dr. Nasr Isfahani

Dr. Nasr Isfahani

ConjugationConjugation

Dr. Nasr Isfahani

Conjugation – A type of gene transfer Conjugation – A type of gene transfer requiring cell-to-cell contactrequiring cell-to-cell contact

Dr. Nasr Isfahani

The F plasmid and conjugation

Dr. Nasr Isfahani

F plasmid

• About 100 gene• Replication• Conjucation• F++→ F++?→ F++?• F+ → F-?F+ → F-?• Sometime: one or more IS: Hfr, FSometime: one or more IS: Hfr, F΄́

• Dr. Nasr Esfahani

Dr. Nasr Isfahani

Dr. Nasr Isfahani

plasmids that can plasmids that can integrate into host integrate into host

chromosomechromosome

Dr. Nasr Isfahani

F+ Hfr

Dr. Nasr Esfahani

Gene transfer in a mating between Hfr donor and F- recipient

Dr. Nasr Isfahani

Dr. Nasr Esfahani

• The rate of chromosomal transfer from Hfr cells is constant,

• results from many conjugation experiments has allowed preparation of an E coli genetic map in which distances between loci are measured in number of minutes required for transfer in conjugation.

Dr. Nasr Isfahani

Dr. Nasr Isfahani

F (fertility) primeF (fertility) prime (F’)

• Integration of chromosomal DNA into a conjugal plasmid can produce a recombinant replicon—F (fertility) prime, or R (resistance) prime

Dr. Nasr Isfahani

F (fertility) prime F (fertility) prime (F’) , or R (resistance) prime , or R (resistance) prime (R’)

Dr. Nasr Isfahani

Hfr F’

(F’)

Dr. Nasr Esfahani

◦Characteristics of F’ x F- crossesF- becomes F’ while F’ remains F’

partial diploids, or merodiploids,

Bacteria carrying gene copies, a full set on the chromosome and a partial set on a prime,

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Transduction

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Transduction

Dr. Nasr IsfahaniFig. 14.19

Dr. Nasr Isfahani

• Direct uptake of donor DNA by recipient cells,

• depends on their competence for transformation.

Dr. Nasr Isfahani

TransformationTransformation

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Mutations are changes in DNA sequence. Spontaneous mutations for a given gene

generally occur with a frequency of 10–8–10–6 in a population derived from a single bacterium.

The mutations includebase substitutions, deletions, insertions, and rearrangements.

Dr. Nasr Isfahani

Bacterial mutantsBacterial mutants

Base substitutions Base substitutions

can arise as a consequence of mispairing between complementary bases during replication.

In E coli, this occurs about once every 1010 times it incorporates a nucleotide

Dr. Nasr Isfahani

Bacterial mutantsBacterial mutants

The base substitution: - missense mutation: point mutation, a

single nucleotide is changed: codon codes different aa

- nonsense mutation: point mutation, premature stop codon

Frame shift mutation: wrong aa downstream

Dr. Nasr Isfahani

Frameshift mutations:introduction or removal of a

single base pair from DNA—are caused by slight slippage of DNA strands.

This slippage is favored by acridine dyes, which can intercalate between bases.

Dr. Nasr Isfahani

Mutagens Mutagens

Dr. Nasr Isfahani

Dr. Nasr Isfahani

3. Viruses: - mu,

- frame shift or deletion

Dr. Nasr Isfahani

Gene expressionGene expression

Dr. Nasr Isfahani

Protein synthesis: Translation initiationProtein synthesis: Translation initiation

Dr. Nasr Isfahani

Protein synthesis:Protein synthesis:elongation (2)elongation (2)

Dr. Nasr Isfahani

Protein Protein translation:translation:termination (3)termination (3)

Dr. Nasr Isfahani

Protein translation: Protein translation: summarysummary

Initiation

Elongation

Termination

http://www.phschool.com/science/biology_place/biocoach/translation/init.html

Dr. Nasr Isfahani

Regulation of Gene expressionRegulation of Gene expression

Dr. Nasr Isfahani

Genetic Regulatory Genetic Regulatory MechanismsMechanisms

Dr. Nasr Isfahani

Transcriptional control

Dr. Nasr Isfahani

Control of Gene ExpressionControl of Gene Expression

Clustering of genes with related function

Coordinate control of genes with related function

Polycistronic mRNA

Dr. Nasr Isfahani

Inducible Genes - Operon Inducible Genes - Operon ModelModel

Definition: Genes whose expression is turned on by the presence of some substance◦Lactose induces expression of the lac genes

◦An antibiotic induces the expression of a resistance gene

Catabolic pathways

Definition: Genes whose expression is turned on by the presence of some substance◦Lactose induces expression of the lac genes

◦An antibiotic induces the expression of a resistance gene

Catabolic pathways

Dr. Nasr Isfahani

Lactose OperonLactose Operon

Structural genes◦lac z, ◦lac y, & ◦lac a◦Promoter◦Polycistronic

Dr. Nasr Isfahani

i

Operon

Regulatory Gene

p o z y a DNA

m-RNA

-GalactosidasePermease

Transacetylase

Protein

Lactose OperonLactose Operon

Inducer : lactose◦Absence

Active repressor No expression

Dr. Nasr Isfahani

i p o z y a

No lac mRNA

Absence of lactose

Active

i p o z y a

-Galactosidase Permease Transacetylase

Presence of lactose

Inactive

– Presence

• Inactivation of repressor

• Expression

Mechanism of Catabolite RepressionMechanism of Catabolite Repression

Dr. Nasr Isfahani

Absence of glucose

i p o z y a

Active

-Galactosidase Permease Transacetylase

CAP

Inactive by Lac

ATPAdenyl cyclase

c-AMP

Maximum expression

Mechanism of Catabolite RepressionMechanism of Catabolite Repression Mechanism of Catabolite RepressionMechanism of Catabolite Repression

Glucose:cAMPCAP (CRP)

proteinNo CAP-cAMP

complex◦No Promoter

activation

Dr. Nasr Isfahani

Presence of glucose

i p o z y a

Inactive

ATPAdenyl cyclase

CAP X

-Galactosidase Permease Transacetylase

Low level expression

Genetic EngineeringGenetic EngineeringGenetic EngineeringGenetic Engineering

Dr. Nasr Isfahani

DNA Cloning with Plasmid DNA Cloning with Plasmid VectorsVectors

Dr. Nasr Isfahani

Plasmids Are Extrachromosomal Self-Replicating DNA Molecules

E. Coli Plasmids Can Be Engineered for Use as Cloning Vectors

Plasmid DNA ReplicationSelection of Transformed CellsPlasmid Cloning Permits Isolation of DNA

Fragments from Complex MixturesRestriction Enzymes Cut DNA Molecules at

Specific SequencesRestriction Fragments with

Complementary "Sticky Ends" Are Ligated Easily

Polylinkers Facilitate Insertion of Restriction Fragments into Plasmid Vectors

Small DNA Molecules Can Be Chemically Synthesized

SUMMARYDr. Nasr Isfahani

Figure. Diagram of a simple cloning vector derived from a plasmid, a circular, double-stranded DNA molecule that can replicate within an E. coli cell.

Dr. Nasr Isfahani

Plasmid vectors contain - a replication origin (ORI) sequence - a gene that permits selection

-usually by conferring resistance to a particular drug. Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin. Exogenous DNA can be inserted into the bracketed region without disturbing the ability of the plasmid to replicate or express the ampr gene.

Dr. Nasr Isfahani

Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin.

Exogenous DNA can be inserted into the bracketed region without disturbing the ability of the plasmid to replicate or express the ampr gene.

Dr. Nasr Isfahani

Figure 2. Plasmid DNA replication. The parental strands are shown in blue, and newly synthesized daughter strands are shown in red

Dr. Nasr Isfahani

Figure 3. General procedure for cloning a DNA fragment in a plasmid vector.

Dr. Nasr Isfahani

Figure 4. Isolation of DNA fragments from a mixture by cloning in a plasmid vector

Dr. Nasr Isfahani

Figure 5. Restriction-recognition sites are short DNA sequences recognized and cleaved by various restriction endonucleases.

Dr. Nasr Isfahani

Figure 6. Fragments produced by cleavage of the ≈36-kb DNA genome from adenovirus 2 (Ad2) by EcoRI and another restriction enzyme, HindIII from Haemophilus influenzae.

Dr. Nasr Isfahani

Figure 7-7. Ligation of restriction fragments with complementary sticky ends.

Dr. Nasr Isfahani

Figure 8. Plasmid vectors containing a polylinker, or multiple-cloning-site sequence, commonly are used to produce recombinant plasmids carrying exogenous DNA fragments.

Dr. Nasr Isfahani

Figure 7-9. Chemical synthesis of oligonucleotides by sequential addition of reactive nucleotide derivatives in the 3 → 5 direction.

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Plasmid vectors

AdvantageRelative simplicity

Disadvantages- Low efficiency of transformation

-Small number of colonies that can be grown on plate.

(1.5 × 105 clones carrying 20-kb DNA for human haploid genome)

Bacteriophages

Larger DNA Fragments Can Be Cloned in Cosmids and Other Vectors

Dr. Nasr Isfahani

Both lambda phage vectors and the more commonly used E. coli plasmid vectors are useful for cloning DNA fragments up to ≈20 - 25 kb.

cosmid cloning up to 45 kb

Dr. Nasr Isfahani

Dr. Nasr Isfahani

Figure 7-16. General procedure for cloning DNA fragments in cosmid vectors. This procedure has the high efficiency associated with phage cloning and permits cloning of restriction fragments up to  45 kb long. In this example, four different types of recombinant cosmid virions could be generated, each carrying one of the genomic fragments indicated by different colors. Plating of the recombinant virions on E. coli cells would yield four different types of colonies, but only one is depicted. Note that the lengths of vector DNA and genomic fragments are not to scale. See text for further discussion.

Dr. Nasr Isfahani

Figure 7-17. Membrane-hybridization assay for detecting nucleic acids. This assay can be used to detect both DNA and RNA, and the radiolabeled complementary probe can be either DNA or RNA

Dr. Nasr Isfahani

Maxam-Gilbert method for sequencing DNA Maxam-Gilbert method for sequencing DNA fragments up to ≈500 nucleotides in lengthfragments up to ≈500 nucleotides in length. . The double-stranded fragment to be sequenced is labeled at the 5 The double-stranded fragment to be sequenced is labeled at the 5 ends with 32P. The label (red circle) is removed from one end, and ends with 32P. The label (red circle) is removed from one end, and the fragment then is denatured. Four identical samples of the the fragment then is denatured. Four identical samples of the prepared fragment are subjected to four different sets of chemical prepared fragment are subjected to four different sets of chemical reactions that selectively cut the DNA backbone at G, G + A, C + T, reactions that selectively cut the DNA backbone at G, G + A, C + T, or C residues. The reactions are controlled so that each labeled or C residues. The reactions are controlled so that each labeled chain is likely to be broken only once. The labeled subfragments chain is likely to be broken only once. The labeled subfragments created by all four reactions have the label at one end and the created by all four reactions have the label at one end and the chemical cleavage point at the other. Gel electrophoresis and chemical cleavage point at the other. Gel electrophoresis and autoradiography of each separate mixture yield one radioactive autoradiography of each separate mixture yield one radioactive band for each nucleotide in the original fragment, each separated band for each nucleotide in the original fragment, each separated according to their length. Bands appearing in the G and C lanes can according to their length. Bands appearing in the G and C lanes can be read directly. Bands in the A + G lane that are not duplicated in be read directly. Bands in the A + G lane that are not duplicated in the G lane are read as A. Bands in the T + C lane that are not the G lane are read as A. Bands in the T + C lane that are not duplicated in the C lane are read as T. The sequence is read from the duplicated in the C lane are read as T. The sequence is read from the bottom of the gel up.bottom of the gel up.

Dr. Nasr Isfahani

Figure 7-28. Figure 7-28. Structures of Structures of

ribonucleoside ribonucleoside triphosphate triphosphate (NTP),(NTP),

deoxyribonucleosiddeoxyribonucleoside triphosphate e triphosphate (dNTP), (dNTP), and and dideoxyribonucleosdideoxyribonucleoside triphosphate ide triphosphate (ddNTP).(ddNTP).

Dr. Nasr Isfahani

Sanger (dideoxy) method for sequencing Sanger (dideoxy) method for sequencing

DNA fragmentsDNA fragments Dr. Nasr Isfahani

DNA sequencingDNA sequencingDr. Nasr Isfahani

• To identify the macromulecules of interest(DNA,RNA OR Protein) among a large number of other molecules.

Dr. Nasr Isfahani

Basic DefinitionsBasic DefinitionsBasic DefinitionsBasic Definitions

Blots:Target molecules

Probes:Complementary sequences of nucleotide bases to the specific RNA or DNA sequence of intrested.

Dr. Nasr Isfahani

The The PolymeraPolymerase Chain se Chain ReactionReaction

(PCR) (PCR)

Dr. Nasr Isfahani

ConjugationConjugation

The F plasmid A) is made of single-stranded DNA B) is present in F- cells C) codes for resistance to antibiotics D) codes for making the F pilus

2 The F pilus A) is a protein appendage on the F+ donor that attaches to specific receptors on the cell wall of the recipient B) is made of nucleotides C) is transferred to the recipient cell D) is a part of the recipient cell that specifically attaches to the donor cell

3 When an F+ donor gives an F plasmid to an F- recipient A) both become F- B) both become F+ C) the donor becomes F- D) the recipient becomes F- E) it is not possible to predict the outcome

4 Contact is required between an F+ and an F- cell for conjugation to occur. A) True B) False

5 When F+ cells are mixed with F- cells, eventually all the cells will become F+. A) True B) False

Dr. Nasr Isfahani

Protein synthesis: Translation Protein synthesis: Translation initiationinitiation

Dr. Nasr Isfahani

Protein Protein synthesis:synthesis:elongation (2)elongation (2)

Dr. Nasr Isfahani

Protein Protein translationtranslation::terminatiotermination (3)n (3)

Dr. Nasr Isfahani

Protein translation: Protein translation: summarysummary

Dr. Nasr Isfahani

Initiation

Elongation

Termination

http://www.phschool.com/science/biology_place/biocoach/translation/init.html