Figure 15.UN03b

TestcrossOffspring

Expected(e)

Observed(o)

Deviation(o − e) (o − e)2 (o − e)2/e

(A−B−)(aaB−)

(A−bb)(aabb)

220210

231239

2 = Sum

Review the Chi-Square Test

Try: 72; 131; 134; 63 for observed

Chapter 16

The Molecular Basis of Inheritance

In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA

The Search for the Genetic Material: Scientific Inquiry

• When Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material

• The key factor in determining the genetic material was choosing appropriate experimental organisms

• The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them

Evidence That DNA Can Transform Bacteria

• The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928

• Griffith worked with two strains of a bacterium, a pathogenic “S” strain and a harmless “R” strain

• When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic

• He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA

Figure 16.2

Living S cells(pathogeniccontrol)

Experiment

Results

Living R cells(nonpathogeniccontrol)

Heat-killed S cells(nonpathogeniccontrol)

Mouse dies Mouse healthy Mouse healthy Mouse dies

Mixture of heat-killed S cells andliving R cells

Living S cells

• In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA

• Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria

• Many biologists remained skeptical, mainly because little was known about DNA

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9-11

Avery et al also conducted the following experiments To further verify that DNA, and not a contaminant (RNA or protein), is the

genetic material

1. Which of the following results from Griffith’s experiment is an example of transformation?

a. Mouse dies after being injected with living S cells.b. Mouse is healthy after being injected with living R cells.c. Mouse is healthy after being injected with heat-killed

S cells.d. Mouse dies after being injected with a mixture of heat-

killed S and living R cells.e. In blood samples from the mouse in “d”, living S cells

were found.

1. Which of the following results from Griffith’s experiment is an example of transformation?

a. Mouse dies after being injected with living S cells.b. Mouse is healthy after being injected with living R cells.c. Mouse is healthy after being injected with heat-killed

S cells.d. Mouse dies after being injected with a mixture of heat-

killed S and living R cells.e. In blood samples from the mouse in “d”, living S cells

were found.

• In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2

• To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection

• They concluded that the injected DNA of the phage provides the genetic information

Figure 16.3

Phagehead

DNA

Tailsheath

Tail fiberGeneticmaterial

Bacterialcell

100

nm

Evidence That Viral DNA Can Program Cells

Figure 16.4

ExperimentBatch 1: Radioactive sulfur (35S) in phage protein

Batch 2: Radioactive phosphorus (32P) in phage DNA

Labeled phagesinfect cells.

Agitation frees outsidephage parts from cells.

Centrifuged cellsform a pellet.

Radioactivity(phage protein)found in liquid

Pellet

Centrifuge

Radioactiveprotein

RadioactiveDNA

Radioactivity (phageDNA) found in pellet

Centrifuge

Pellet

1 2

4

3

4

Additional Evidence That DNA Is the Genetic Material

• In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next

A & T same ratio; G & C same ratio, but % GC varies from species to species (Chargaff’s rule)

• This evidence of diversity made DNA a more credible candidate for the genetic material

• By the 1950s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group

Figure 16.5

5′ end

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

Nitrogenousbases

Sugar–phosphatebackbone

3′ end

Nitrogenousbase

Sugar(deoxyribose)DNA

nucleotide

Phosphate

Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure

Franklin’s X-ray diffractionphotograph of DNA

Rosalind Franklin

• Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical

• The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases

• The width suggested that the DNA molecule was made up of two strands, forming a double helix, anti-parallel in nature

B-DNA2 nm wide; 3.6 angstrom per unit; 10 units per turn

Figure 16.7

(a) Key features ofDNA structure

(b) Partial chemical structure

0.34 nm

3′ end

5′ endTT

T

AA

A

C

CC

GG

G

AT1 nm

TA

C G

CG

AT3.4 nm

CGCG

C GC G

3′ end

5′ end

Hydrogen bond

T A

G C

A T

C G

(c) Space-fillingmodel

• Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA

• Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior

• At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width

• Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray

Figure 16.UN02

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: widthconsistent with X-ray data

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

Sugar

Sugar

Sugar

Sugar

•Watson and Crick reasoned that the pairing was more

specific, dictated by the base structures•They determined

that adenine paired only with thymine, and guanine paired only with cytosine

The Basic Principle: Base Pairing to a Template Strand

• Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication

• In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

•Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material

Figure 16.9-3

(a) Parentalmolecule

(b) Separation of parentalstrands into templates

A

A

A

T

T

T

G

G C

C

A

A

A

T

T

T

G

G C

C

A

A

A

T

T

T

G

G C

C

A

A

A

T

T

T

G

G C

C

(c) Formation of new strandscomplementary to templatestrands

• Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand

• Competing models were the conservative model and the dispersive model

• Meselson and Stahl provided the supporting scientific evidence

Figure 16.10

(a) Conservativemodel

(b) Semiconserva-tive model

(c) Dispersivemodel

Parent cellFirst

replicationSecond

replication

• Experiments by Meselson and Stahl supported the semiconservative model

• They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope

• The first replication produced a band of hybrid DNA, eliminating the conservative model

• A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model

Figure 16.11

Bacteria culturedin medium with 15N(heavy isotope)

Experiment

Results

Conclusion

Bacteria transferredto medium with 14N(lighter isotope)

DNA samplecentrifugedafter firstreplication

DNA samplecentrifugedafter secondreplication

Less dense

More dense

Predictions: First replication Second replication

Conservativemodel

Semiconservativemodel

Dispersivemodel

1 2

43

DNA Replication: A Closer Look

• The copying of DNA is remarkable in its speed and accuracy• More than a dozen enzymes and other proteins participate

in DNA replication

Video: DNA Replication

2. What is the %T in wheat DNA?

a. approximately 22%b. approximately 23%c. approximately 28%d. approximately 45%

2. What is the %T in wheat DNA?

a. approximately 22%b. approximately 23%c. approximately 28%d. approximately 45%

Getting Started: Origins of Replication

• Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”

• A eukaryotic chromosome may have hundreds or even thousands of origins of replication

• Replication proceeds in both directions from each origin, until the entire molecule is copied

• At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating

Figure 16.12

Origin of replication

0.5

µm

0.25

µm

Bacterialchromosome

Two daughterDNA molecules

Replicationbubble

Parental (template)strand Daughter

(new) strand

Replicationfork

Double-stranded

DNA molecule

(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cellOrigin of

replication Eukaryotic chromosome

Double-strandedDNA molecule

Parental (template)strand

Daughter (new) strand

Replicationfork

Bubble

Two daughter DNA molecules

Elongating a New DNA Strand

• Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork

• Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate

• The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

LE 16-13

New strand5 end

Phosphate BaseSugar

Template strand3 end 5 end 3 end

5 end

3 end

5 end

3 end

Nucleosidetriphosphate

DNA polymerase

Pyrophosphate

Antiparallel Elongation

• The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication

• DNA polymerases add nucleotides only to the free 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5to3direction

• Along one template strand of DNA, called the leading strand, DNA polymerase can synthesize a complementary strand continuously, moving toward the replication fork

• To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork

• The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase

LE 16-14

Parental DNA

5

3

Leading strand

35

3

5

Okazakifragments

Lagging strand

DNA pol III

Templatestrand

Leading strand Lagging strand

DNA ligase Templatestrand

Overall direction of replication

Priming DNA Synthesis

• DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end

• The initial nucleotide strand is a short one called an RNA or DNA primer

• An enzyme called primase can start an RNA chain from scratch

• Only one primer is needed to synthesize the leading strand, but for the lagging strand each Okazaki fragment must be primed separately

Figure 16.16Overview

12

1

1

21

2

1

1

2

5′

3′

3′

5′5′3′3′

3′ 3′5′5′

5′5′

3′3′

5′3′

5′3′

5′3′

5′3′

5′3′

5′3′

1

2 5

6

4

3

Origin of replicationLaggingstrand

Leadingstrand

Laggingstrand

LeadingstrandOverall directions

of replication

RNA primerfor fragment 2

Okazakifragment 2

DNA pol IIImakes Okazakifragment 2.

DNA pol Ireplaces RNAwith DNA.

DNA ligaseforms bondsbetween DNAfragments.

Overall direction of replication

Okazakifragment 1

DNA pol IIIdetaches.

RNA primerfor fragment 1

Templatestrand

DNA pol IIImakes Okazakifragment 1.

Origin ofreplication

Primase makesRNA primer.

5′

Other Proteins That Assist DNA Replication

• Helicase untwists the double helix and separates the template DNA strands at the replication fork

• Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template

• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

• Primase synthesizes an RNA primer at the 5 ends of the leading strand and the Okazaki fragments

• DNA pol III continuously synthesizes the leading strand and elongates Okazaki fragments

• DNA pol I removes primer from the 5 ends of the leading strand and Okazaki fragments, replacing primer with DNA and adding to adjacent 3 ends

• DNA ligase joins the 3 end of the DNA that replaces the primer to the rest of the leading strand and also joins the lagging strand fragments

Figure 16.17

Overview

5′

3′

Laggingstrand

Leadingstrand

Leadingstrand

Laggingstrand

Leading strand

Leading strandtemplate

Origin of replication

Overall directionsof replication

5′

3′5′ 3′

5′

5′

3′3′

3′

Single-strandbinding proteins

Helicase

ParentalDNA

DNA pol IIIPrimer

PrimaseLaggingstrand

Lagging strandtemplate

DNA pol IIIDNA pol I

5′DNA ligase

1234

5

The DNA Replication Machine as a Stationary Complex

• The proteins that participate in DNA replication form a large complex, a DNA replication “machine”

Called the Replisome

• The DNA replication machine is probably stationary during the replication process

• Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules

Table 16.1

Proofreading and Repairing DNA

• DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides

• In mismatch repair of DNA, repair enzymes correct errors in base pairing

• In nucleotide excision repair, enzymes cut out and replace damaged stretches of DNA

LE 16-17

DNA ligase

DNA polymerase

DNA ligase seals thefree end of the new DNAto the old DNA, making thestrand complete.

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

A nuclease enzyme cutsthe damaged DNA strandat two points and the damaged section isremoved.Nuclease

A thymine dimerdistorts the DNA molecule.

Replicating the Ends of DNA Molecules• Limitations of DNA polymerase create problems for

the linear DNA of eukaryotic chromosomes• The usual replication machinery provides no way to

complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules

Figure 16.20

Ends of parentalDNA strands

Lagging strand

Parental strand

RNA primer

Last fragmentNext-to-last

fragment

Lagging strandLeading strand

Removal of primers andreplacement with DNAwhere a 3′ end is available

3′5′

5′

Second roundof replication

Further roundsof replication

New lagging strand

New leading strand

Shorter and shorter daughter molecules

5′3′

3′

5′3′

5′

3′

• Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres

• Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules

• It has been proposed that the shortening of telomeres is connected to aging

Figure 16.21

1 µm

• If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce

• An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells using an enzyme with an RNA template

Concept 16.3: A chromosome consists of a DNA molecule packed

together with proteins• The bacterial chromosome is a double-

stranded, circular DNA molecule associated with a small amount of protein

• Eukaryotic chromosomes have linear DNA molecules associated with a large amountof protein

• In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid

• In the eukaryotic cell, DNA is precisely combined with proteins in a complex called chromatin

• Chromosomes fit into the nucleus through an elaborate, multilevel system of packing

Figure 16.22

DNA, thedouble helix

Histones Nucleosomes,or “beads ona string”(10-nm fiber)

30-nm fiberLoopeddomains(300-nm fiber)

Metaphasechromosome

DNAdouble helix(2 nm in diameter)

Nucleosome(10 nm in diameter)

30-nm fiber

Loops Scaffold

Histones Histone tailH1

300-nmfiber

Chromatid(700 nm)

Replicatedchromosome(1,400 nm)

Figure 16.23

5 µm

• Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis

• Loosely packed chromatin is called euchromatin• During interphase a few regions of chromatin

(centromeres and telomeres) are highly condensed into heterochromatin

• Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions

3. Which of the following statements is false when comparing prokaryotes with eukaryotes?

A) The prokaryotic chromosome is circular, whereas eukaryotic chromosomes are linea

B) Prokaryotic chromosomes have a single origin of replication, whereas eukaryotic chromosomes have many.

C) The rate of elongation during DNA replication is higher in prokaryotes than in eukaryotes.

D) Prokaryotes produce Okazaki fragments during DNA replication, but eukaryotes do not.

E) Eukaryotes have telomeres, and prokaryotes do not.

3. Which of the following statements is false when comparing prokaryotes with eukaryotes?

A) The prokaryotic chromosome is circular, whereas eukaryotic chromosomes are linea

B) Prokaryotic chromosomes have a single origin of replication, whereas eukaryotic chromosomes have many.

C) The rate of elongation during DNA replication is higher in prokaryotes than in eukaryotes.

D) Prokaryotes produce Okazaki fragments during DNA replication, but eukaryotes do not.

E) Eukaryotes have telomeres, and prokaryotes do not.