Pierce Genetics: A Conceptual Approach 3ebstephen.weebly.com/uploads/7/8/8/1/7881286/ch_8_9... · GENETICS ESSENTIALS Concepts and Connections THIRD EDITION RNA, a Single Strand of

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CHAPTER 8

DNA: The Chemical Nature of the GeneKey Characteristics of Genetic Material

• Genetic material must contain complex information

• Genetic material must replicate faithfully

• But also must have the capacity to vary

• Genetic material must encode the phenotype.

All Genetic Information Is Encoded in

the Structure of DNA or RNA

• Early studies of DNA

• Miescher: Nuclein

• Kossel: DNA contains four nitrogenous bases

• Chargaff’s rules

• DNA as the source of genetic information

– Identification of the transforming principle

• Griffith experiment

• The experiment by Avery, Macleod, and McCarty

• The Hershey–Chase experiment

Chargaff’s

rules

Griffith

Transforming principle

Avery, McCleod, McCarty

• Treat with enzymes

• DNase

• RNase

• Protease

DNA is transforming principle

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Hershey, Chase

• DNA and Protein w/ radioactive

isotopes

• Phosphorus on DNA

• Sulfur on protein

Hershey, Chase

• DNA and Protein w/ radioactive

isotopes

• Phosphorus on DNA

• Sulfur on protein

• Bacteria w protein coat

removed were radioactive

• So were some of progeny

• DNA is the genetic material

Genetic Information Is Encoded in the

Structure of DNA or RNA

• DNA as the source of genetic information

– Watson and Crick’s discovery of the three-

dimensional structure of DNA

– X-ray diffraction image of DNA

DNA Consists of Two Complementary and

Antiparallel Nucleotide Strands that Form

a Double Helix

• The primary structure of DNA

– Deoxyribonucleotides

• Nucleotides

– Three parts: sugar, a phosphate, and a base

» Purine or pyrimidine base:

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Concept Check

How do the sugars of RNA and DNA differ?

a. RNA has a six-carbon sugar; DNA has a five-

carbon sugar

b. The sugar of RNA has a hydroxyl group that is not

found in the sugar of DNA

c. RNA contains uracil; DNA contain thymine

d. DNA’s sugar has a phosphorus atom; RNA’s sugar

does not



a Double Helix

• Secondary structures of DNA

– The double helix

– Backbone formed through phosphodiester

bonds

– Hydrogen bond and base pairing

– Antiparallel complementary DNA strands

DNA and RNA

DNA

• Sugar/phosphate backbone

• Double stranded

• One Fewer O on sugar

• Strands run antiparallel

• 5’ (5’-carbon to free phosphate)

• 3’ (3’-carbon to free OH)

• Complimentary strands

• Bound by Hydrogen bonds

• T-A (2 bonds)

• C-G (3 bonds)

• Helical

RNA

• Sugar/phosphate backbone

• Single stranded

• Often folds over on self

• OH on sugar

Concept Check

The antiparallel nature of DNA refers to

a. its charged phosphate groups.

b. the pairing of bases on one strand with bases on

the other strand.

c. the formation of hydrogen bonds between bases

from opposite strands.

d. the opposite direction of the two strands of

nucleotides.

• Secondary structures of DNA

– Three-dimensional structure identified by

Watson and Crick refers to B-DNA

– Different secondary structures



a Double Helix

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Large Amounts of DNA Are Packed

into a Cell

• DNA must be tightly packed to fit in small

spaces

• Supercoiling

– Positive supercoiling,

– Negative supercoiling

– Topoisomerase: The enzyme responsible for

adding and removing turns in the coil

The Bacterial Chromosome

• Most bacterial genomes have a single

circular DNA molecule

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Chromatin – Eukaryotic organisms

Chromatin - DNA and Proteins

• Euchromatin

• active

• Heterochromatin

• Inactive (always wrapped)

• Histones – most common protein in chromatin

• DNA wrapped around histones

• Positively charged - attracts Negative DNA

• Nucleosome – beads on a string

• DNA wrapped around 8 histones (2 of each 2A,2B, 3 and 4)

• H1 – acts as spacer (maybe does more)

• Fibers – formed by packing nucleosomes together

DNA Packed into a Cell

Beads attached to about 200 bp

• Chromatin structure

– Changes in chromatin structure

• Epigenetic changes: methylation; capable of being

reversed and often due to environmental factors

DNA Packed into a Cell

Epigenetic

changes: DNA

methylation

causes different

coat colors in

mice

Eukaryotic Chromosomes Possess

Centromeres and Telomeres

• Centromeres

• Constricted portion

• Binding site of

kinetochore (site of

spindle attachment)

• Tandem repeats

• Telomeres

• End of chromosome

• Repeated units

• CCC and GGGs

• Looped over

• Protection

Eukaryotic DNA Contains Several

Classes of Sequence Variation

• Organisms differ in amount of DNA per

cell (C value)

Types of DNA sequences in eukaryotes

– Unique sequence DNA

• Genes that encode proteins

• Gene family: Similar but not identical copies of unique DNA

sequences that arose through duplication of an existing gene

– Repetitive DNA

• Moderately repetitive DNA: 150 ~ 300 bp long

– Tandem repeat sequences

– Interspersed repeat sequences

– Highly Repetitive DNA

• Highly repetitive DNA: less than 10 bp long

– Microsatellite DNA

Eukaryotic DNA Contains Several

Classes of Sequence Variation

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Concept Check

Most of the genes that encode proteins are

found in

a. unique-sequence DNA.

b. moderately repetitive DNA.

c. highly repetitive DNA.

d. All of the above.

GENETICS ESSENTIALS

Concepts and ConnectionsTHIRD EDITION

Benjamin A. Pierce

CHAPTER 9

DNA Replication and Recombination

© 2014 W. H. Freeman and Company

• Replication has to be accurate

– One error per million bp leads to 6400

mistakes every time a cell divides, which

would be catastrophic

• Replication also takes place at high speed

– E. coli replicates its DNA at a rate of 1000

nucleotides per second

Genetic Information Must Be Accurately

Copied Every Time a Cell Divides

DNA Replication Takes Place in a

Semiconservative Manner

• Proposed DNA Replication Models

– Conservative replication model

– Dispersive replication model

– Semiconservative replication

• Meselson and Stahl’s Experiment

– Two isotopes of nitrogen

• 14N common form; 15N rare heavy form

• E.coli were grown in a 15N media first, then

transferred to 14N media

• Cultured E.coli were subjected to equilibrium

density gradient centrifugation



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• Modes of Replication

– Replicons: Units of replication

• Replication origin

– Theta replication: circular DNA, E. coli; single

origin of replication forming a replication fork,

and it is usually a bidirectional replication



Linear eukaryotic replication

– Eukaryotic cells

– Multiple origins - replicons

– A typical replicon: ~ 200,000-300,000 bp in

length.

• Requirements of replication• A template strand

• Raw material: nucleotides

• Enzymes and other proteins



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• Direction of Replication

– DNA polymerase add nucleotides only to the

3 end of growing strand

– The replication can only go from 5 3

– Continuous and discontinuous replication

DNA Replication Cont.


• Direction of replication

• Leading strand: undergoes continuous replication

• Lagging strand: undergoes discontinuous

replication

• Okazaki fragment: the discontinuously synthesized

short DNA fragments forming the lagging strand

DNA Replication Cont.

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Concept Check 2

Discontinuous replication is a result of

which property of DNA?

a. Complementary bases

b. Antiparallel nucleotide strands

c. A charged phosphate group

d. Five-carbon sugar

Bacterial Replication Requires a Large

Number of Enzymes and Proteins

• Bacterial DNA Replication

– Initiation

• oriC (single origin replicon)

• an initiation protein (DnaA in E.coli)

– Unwinding

• DNA helicase

• Single-strand-binding proteins (SSBs)

• DNA gyrase (topoisomerase)

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Replication Cont.

Order used in the course of replication:

Initiator proteins – bind to origin of replication

Helicase - unwinds

Single-strand-binding protein - detach

DNA gyrase – releases stress on molecule

Then elongation

• Elongation

– Primers: an existing group of RNA nucleotides

with a 3-OH group to which a new nucleotide

can be added. It is usually 10 to 12

nucleotides long.

– Primase: RNA polymerase

Bacterial Replication Requires a Large

Number of Enzymes and Proteins

• Elongation: carried out by DNA polymerase III

• Removing RNA primer: DNA polymerase I

‒ Connecting nicks after RNA primers are removed:

DNA ligase

• Termination: when two replication forks meet

or by a specific termination sequence

Replication Cont.

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Bacterial

polymerasesDNA ligase links

Okazaki fragments

Video: 0903

Looping:

• Leading strand and lagging

strand with DNA polymerase

complex

The fidelity of DNA replication

• Errors every 100,000 nucleotides

• Proofreading: DNA polymerase I : 3 5

exonuclease activity removes the incorrectly paired

nucleotide, reduces errors to 1 in 10 million

• Mismatch repair: corrects errors after replication is

complete (more later): Errors reduced even more

Replication Cont.

• Eukaryotic DNA Replication

– Origin-recognition complex (ORC) binds to

origins and initiates DNA replication

• Eukaryotic DNA polymerases

• Replication at the ends of chromosomes‒ Telomeres and telomerase

Eukaryotic DNA Replication Is Similar to

Bacterial Replication but Differs in

Several Aspects

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Many DNA polymerases in eukaryotic cells

Important details

• Multiple origins

• Origin complex

• ORC

• RNA Primers

• DNA nucleotides

• Gap – no opposite

strand

Gap filled, or partially

filled with the help of

telomerase

Gap filled, or partially

filled with the help of

telomerase

Recombination Takes Place Through the

Breakage, Alignment, and Repair of DNA

Strands

• Homologous recombination: exchange is

between homologous DNA molecules

during crossover

• Holliday junction and single-strand break

• The double-strand break model of

recombination

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Death cap mushroom: kills by inhibiting RNA polymerase II

CHAPTER 10

Transcription

GENETICS ESSENTIALS

Concepts and Connections

THIRD EDITION

RNA, a Single Strand of Ribonucleotides,

Participates in a Variety of Cellular Functions

• RNA: evidence suggests RNA was original

genetic material

• The structure of RNA

– Primary structure

– Secondary structure

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Transcription Is the Synthesis of an RNA

Molecule from a DNA Template

The template

– The transcribed strand: template strand

– The transcription unit

• A promoter

• RNA-coding sequence

• Terminator

Sequence forms from one side – template strand – but different

genes may have opposite template strands. Terminology:

sometimes sense strand(+) anti-sense strand (-).

Transcriptional Unit – RNA and other regions

• Promoter – binding site starting transcription

• Coding region - RNA sequence from here

• Terminator – Signal that ends transcription

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The substrate for transcriptionRibonucleoside triphosphates—rNTPs added to the 3 end of the RNA molecule

Transcription Is the Synthesis of RNA

Molecule from a DNA Template

To Know:

Initiation

– The substrate for transcription:

Ribonucleoside triphosphates—rNTPs added

to the 3 end of the RNA molecule

– The transcription apparatus: Eukaryotic RNA

polymerases

Transcription Consists of Initiation, Elongation,

and Termination


and Termination

Initiation

• Bacterial promoters

Consensus sequences: sequences that possess

considerable similarity.

• –10 consensus: 10 bp upstream of the start site

• Pribnow box:

– 5 TATAAT 3

– 3 ATATTA 5

• –35 consensus sequence: TTGACA

Consensus sequence

similar enough so

binding molecule can

bind.

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• Initiation

– Initial RNA synthesis: no primer is required

– The location of the consensus sequence

determines the position of the start site

• Elongation

– RNA elongation is carried out by the action of

RNA polymerase

Transcription Consists of Initiation,

Elongation, and Termination

Holoenzyme is just a term for molecule formed by enzyme and co-enzyme

Concept Check

What is the function of the sigma factor?

The sigma factor controls the binding of RNA

polymerase to the promoter by forming the

holoenzyme.

• Elongation: the molecular structure of

eukaryotic RNA polymerase II and how it

functions during elongation has been revealed

through the work of Roger Kornberg and his

colleagues

• Termination:

– RNA polymerase I

– RNA polymerase II

– RNA polymerase III


and Termination

Termination

– Rho-dependant termination: uses rho factor

– Rho-independent termination: hairpin

structure formed by inverted repeats, followed

by a string of uracils

Transcription Consists of Initiation,

Elongation, and Termination

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RNA termination

• Nucleotides are added to the RNA molecule until it transcribes

a terminator

• Activity mostly known from bacteria

• Rho-dependent

• Inverted sequence forms hairpin

• Poly Us at end cause pause

• Rho protein unwinds DNA/RNA

• Polycistronic (uncommon in eukaryotes)

• Single terminator for several genes

• Will not test on the details of termination

Many Genes Have Complex Structures

• Gene Organization• The concept of colinearity and noncolinearity

• Number of nucleotides in a gene should be

proportional to the number of amino acids in the

encoded protein

• DNA much longer than mRNA; demonstrated through

hybridization

1958 idea - Crick

• DNA sequence

corresponds to protein

sequence

• Colinear

• But DNA is much longer

than the RNA is produces

Collinearity is not the case

Loops when DNA is re-annealed with

complementary RNA indicates nucleotides

not paired

Non-coding region of DNA = loops

Exons = coding

Introns = non-coding

This concept is important

Many Genes Have Complex Structures

•The concept of the gene revisited

•The gene includes:

• DNA sequences that code for all exons and

introns

• Those sequences at the beginning and end of

the RNA that are not translated into a protein,

including the entire transcription unit– The promoter

– The RNA coding sequence

– The terminator

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Many RNA Molecules Are Modified After

Transcription in Eukaryotes

• A mature mRNA contains 5′ untranslated region (5′ UTR, or leader sequence)

◦ Shine–Dalgarno sequence

• Protein-coding region

• 3′ untranslated region



• The addition of the 5′ cap

‒ A nucleotide with 7-methylguanine; 5′-5′ bond is

attached to the 5′-end of the RNA

• The addition of the poly(A) tail

‒ ~ 50 to 250 adenine nucleotides are added to the

3′-end of the mRNA



• RNA splicing

‒ Consensus sequences

• 5′ consensus sequence: GU A/G AGU: 5′ splice

site

• 3′ consensus sequence: CAGG

‒ The process of splicing

RNA processing

• mRNA modified before translation to amino acids

• Cleavage cuts out introns

• Adenine tail added

Ends of exons spliced together

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RNA processing

• 5’ cap – more later

• Poly A tail – slows degradation of RNA

Transfer RNA (tRNA)

• The structure and processing of transfer

RNA (tRNA)

• Common secondary structure—the

cloverleaf structure

• Anticodon

Ribosomal RNA (rRNA) and the Ribosome

• The structure of the ribosome

– Large ribosome subunit

– Small ribosome subunit

Regulation via RNAs

MicroRNAs

• Inhibit translation

Small interfering RNAs

• Degrade mRNA

GENETICS ESSENTIALS


Benjamin A. Pierce

CHAPTER 11

From DNA to Proteins: Translation


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The Genetic Code Determines How the Nucleotide

Sequence Specifies the Amino Acid Sequence of a

Protein

• Breaking the genetic code

• The reading frame and initiation codons

• Termination codons

• The universality of the code

• Breaking the genetic code

• Codon: a triplet RNA code

• 64 possible codons‒ 3 stop codons

‒ 61 sense codons

The Genetic Code

• Synonymous codons: codons that specify the same amino acid

• Isoaccepting tRNAs: different tRNAs that accept the same amino acid but have different anticodons

• Codons

• Sense codons: encoding amino acid

• Initiation codon: AUG

• Termination codon: UAA, UAG, UGA

• Wobble hypothesis

The Genetic Code

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Concept Check

Through wobble, a single can pair with more

than one .

a. codon; anticodon

b. group of three nucleotides in DNA; codon in

mRNA

c. tRNA; amino acid

d. anticodon; codon

•The Reading Frame and Initiation

Codons• Reading frame: Read in groups of three so the

starting point is very important.

• Nonoverlapping: A single nucleotide may not be

included in more than one codon.

• The universality of the code: near universal, with

some exceptions

11.1 The Genetic Code Determines How the

Nucleotide Sequence Specifies the Amino Acid

Sequence of a Protein

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Amino Acids Are Assembled Into a

Protein Through Translation

• The binding of amino acids to transfer RNAs

• The initiation of translation

• Elongation

• Termination

• Amino acids bind to tRNAs• The specificity between an amino acid and its tRNA is

determined by each individual aminoacyl-tRNA synthesis

• There are 20 different aminoacyl-tRNA syntheses in a cell

• The initiation of translation

• Initiation factors IF-3, initiator tRNA with N-

formylmethionine attached to form fmet-tRNA

• Energy molecule: GTP

Translation

•Amino acids bind to tRNAs•The specificity between an amino acid and its

tRNA is determined by each individual

aminoacyl-tRNA synthesis

•There are 20 different aminoacyl-tRNA

syntheses in a cell

• The Initiation of Translation

• The Shine–Dalgarno consensus sequence in

bacterial cells is recognized by the small unit

of ribosome.

• The Kozak sequence in eukaryotic cells

facilitates the identification of the start codon.

11.2 Amino Acids Are Assembled into a

Protein Through Translation

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• Elongation

• Exit site E

• Peptidyl site P

• Aminoacyl site A

• Elongation factors: Tu, Ts, and G

Translation

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• Termination

• Termination codons: UAA, UAG, and UGA

• Release factors (RFs)

Translation

Mini video

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• Polyribosomes: an mRNA with several

ribosomes attached

Additional Properties

• Proteins go through modifications after

translation

• Folding

• Most proteins must fold into a 3D shape in order to

be functional

• Molecular chaperones

• Molecules that prevent incorrect folding

Additional Properties, Proteins

GENETICS ESSENTIALS


Benjamin A. Pierce

CHAPTER 14

Molecular Genetic Analysis and

Biotechnology


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Techniques of Molecular Genetics Have

Revolutionized Biology

• Recombinant DNA Technology (Genetic Engineering)– Techniques for locating, isolating, altering, and

studying DNA segments

• The Molecular Genetics Revolution– Biotechnology: the use of these techniques to

develop new products

• Working at the Molecular Level

Molecular Techniques Are Used to Isolate,

Recombine, and Amplify Genes

• First step: isolate DNA segment or gene

• Cutting and joining DNA fragments—restriction

enzymes

• Viewing DNA fragments

• Locating DNA fragments with Southern blotting

and probes

Cutting and Joining DNA

Fragments

• Restriction enzymes: recognizing and cutting

DNA at specific nucleotide sequences

• Type II restriction enzyme: most useful enzyme

• By adding methyl groups to the recognition

sequence to protect itself from being digested by

its own enzyme in bacteria

Cutting and Joining DNA

Fragments

• Cohesive ends: fragments with short, single-

stranded overhanging ends

• Blunt ends: even-length ends from both single

strands

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Viewing DNA

Fragments• Gel electrophoresis

• Autoradiography

Concept Check

DNA fragments that are 500 bp, 1000 bp, and

2000 bp in length are separated by gel

electrophoresis. Which fragment will migrate

farthest in the gel?

a. The 2000-bp fragment.

b. T he 1000-bp fragment.

c. The 500-bp fragment.

d. All fragments will migrate equal distances.

Locating DNA Fragments with

Southern Blotting and Probes

• Probe: DNA or RNA with a base sequence

complementary to a sequence in the gene of

interest

Southern blot named after Edwin Southern, similar methods named after take

off on directional aspect: Western blot, Northern Blot, Eastern blot.

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Molecular Techniques Are Used to Isolate,

Recombine, and Amplify Genes

• Cloning genes

• Application: the genetic engineering of plants

with pesticides

• Amplifying DNA fragments with the polymerase

chain reaction

Cloning Genes

• Gene cloning: amplifying a specific piece of

DNA via a bacteria cell

• Cloning vector: a replicating DNA molecule

attached with a foreign DNA fragment to be

introduced into a cell

Cloning Genes

• Plasmid vectors

– Plasmids: circular DNA molecules from bacteria

– Insert foreign DNA into plasmid using restriction

enzymes

– Linkers: synthetic DNA fragments containing

restriction sites

• Transformation of host cells with plasmids

• Screening cells for recombinant plasmids

– Selectable markers are used to confirm whether the

cells have been transformed

Cloning Genes

• Other gene vectors:

– Cosmids

– Bacterial artificial chromosomes (BACs)

– Yeast artificial chromosome

– Plasmid

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Concept Check 3

How is a gene inserted into a plasmid cloning

vector?

The gene and plasmid are cut with the same

restriction enzyme and mixed together. DNA ligase

is used to seal nicks in the sugar–phosphate

backbone.

Amplifying DNA Fragments with the

Polymerase Chain Reaction (PCR)

• The PCR reaction

– Taq polymerase: stable DNA polymerase at high

temperature

– Reverse-transcription PCR

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• Limitations of PCR

– Prior knowledge of target DNA

– Contamination

– Accuracy

– Amplified fragments are less than 2 kb



• Applications of PCR

– Real-time PCR: quantitatively determining the

amount of DNA amplified as the reaction proceeds

Molecular Techniques Can Be Used to Find

Genes of Interest

• Gene libraries

• In situ hybridization

• Positional cloning

• Application: isolating the gene for cystic fibrosis

Gene Libraries

• DNA library: a collection of clones containing all the DNA fragments from one source

– Creating a genomic DNA library

– cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA

Gene Libraries

• Screening DNA libraries

– Plating clones of the library

– Probing plated colonies or plaques

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Positional Cloning

• Isolating genes on the basis of their position on

a genetic map

• Chromosome walking

• Chromosome jumping• Larger steps “jump” around

14.3 DNA Sequences Can Be Determined

and Analyzed

• Restriction Fragment Length Polymorphisms

(RFLPs)

• DNA sequencing

• Next-generation sequencing technologies

• DNA fingerprinting

Restriction Fragment Length Polymorphisms

• Some DNA fragments have different restriction

sites due to mutation for the same restriction

enzyme

• Causes polymorphisms within a population

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DNA Sequencing

• Sanger’s dideoxy-sequencing method

– Dideoxyribonucleoside triphosphate (ddNTP)

lacks a 3′-oh group, which terminates DNA

synthesis

No 3’-OH group

Terminates synthesis

Concept Check 4

In the dideoxy-sequencing reaction, what

terminates DNA synthesis at a particular base?

a. The absence of a base on the ddNTP halts the

DNA polymerase

b. The ddNTP causes a break in the sugar–phosphate

backbone

c. DNA polymerase will not incorporate a ddNTP into

the growing DNA strand

d. The absence of a 3’-OH group

Next-Generation Sequencing Technologies

• Pyrosequencing

• Illumina sequencing

• Third-generation sequencing

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DNA Fingerprinting (DNA Profiling)

• Microsatellites: (short tandem repeats, STRs)

variable number of copies of repeat sequences

possessed by many organisms

• Detected by PCR

• Fragments represented as peaks on a graph

– Homozygotes: single tall peak

– Heterozygotes: two shorter peaks

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14.4 Molecular Techniques Are Increasingly

Used to Analyze Gene Function

• Forward and reverse genetics

• Transgenic animals

• Knockout mice

Forward and Reverse Genetics

• Forward genetics: Begins with a phenotype to

a gene that encodes the phenotype

• Reverse genetics: Begins with a gene of

unknown function, first inducing mutations

and then checking the effect of the mutation

on the phenotype

Transgenic Animals

• An organism permanently altered by the addition

of a DNA sequence to its genome

• Transgene

Knockout Mice

• A normal gene of the mouse has been fully

disabled

• Knock-in mice: a mouse carries an inserted

DNA sequence at specific locations

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Biotechnology Harnesses the Power

of Molecular Genetics

• Pharmaceutical products

• Specialized bacteria

• Agriculture products

• Genetic testing

• Gene therapy

Potential Benefits of genetically

Engineered plant?• Pest resistant

• Increased yield

• Increased resistance to env. stress – Drought

• Produce product, other than food– Hormone/enzyme

Documents

Pierce Genetics: A Conceptual Approach 3ebstephen.weebly.com/uploads/7/8/8/1/7881286/ch_8_9... · GENETICS ESSENTIALS Concepts and Connections THIRD EDITION RNA, a Single Strand of