Molecular biology 5th ed r weaver (mcgraw hill, 2012)

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Molecular Biology
Fifth Edition

R o b e r t F. We a v e r
University of Kansas

TM


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TM

MOLECULAR BIOLOGY, FIFTH EDITION

Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the
Americas, New York, NY 10020. Copyright © 2012 by The McGraw-Hill Companies, Inc. All rights reserved.
Previous editions © 2008, 2005, and 2002. No part of this publication may be reproduced or distributed in
any form or by any means, or stored in a database or retrieval system, without the prior written consent of
The McGraw-Hill Companies, Inc., including, but not limited to, in any network or other electronic storage or
transmission, or broadcast for distance learning.
Some ancillaries, including electronic and print components, may not be available to customers outside the
United States.
This book is printed on acid-free paper.
1 2 3 4 5 6 7 8 9 0 QDB /QDB 1 0 9 8 7 6 5 4 3 2 1
ISBN 978-0-07-352532-7
MHID 0-07-352532-4
Vice President & Editor-in-Chief: Marty Lange
Vice President EDP/Central Publishing Services: Kimberly Meriwether David
Publisher: Janice Roerig-Blong
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All credits appearing on page or at the end of the book are considered to be an extension of the
copyright page.
Library of Congress Cataloging-in-Publication Data
Weaver, Robert Franklin, 1942Molecular biology / Robert F. Weaver.—5th ed.

p. cm.
ISBN 978–0–07–352532–7 (hardcover : alk. paper)
1. Molecular biology. I. Title.
QH506.W43 2011
572.8—dc22
2010051759

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To Camilla and Nora


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A B O U T

T H E

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A U T H O R

Rob Weaver was born in Topeka, Kansas, and
grew up in Arlington, Virginia. He received his
bachelor’s degree in chemistry from the College

of Wooster in Wooster, Ohio, in 1964. He
earned his Ph.D. in biochemistry at Duke
University in 1969, then spent two years doing
postdoctoral research at the University of
California, San Francisco, where he studied the
structure of eukaryotic RNA polymerases with
William J. Rutter.
He joined the faculty of the University of
Kansas as an assistant professor of biochemistry
in 1971, was promoted to associate professor,
and then to full professor in 1981. In 1984, he
became chair of the Department of Biochemistry,
(Source: Ashvini C. Ganesh)
and served in that capacity until he was named
Associate Dean of the College of Liberal Arts
and Sciences in 1995.
Prof. Weaver is the divisional dean for the science and mathematics departments
within the College, which includes supervising 10 different departments and
programs. As a professor of molecular biosciences, he teaches courses in introductory
molecular biology and the molecular biology of cancer. In his research laboratory,
undergraduates and graduate students have participated in research on the molecular
biology of a baculovirus that infects caterpillars.
Prof. Weaver is the author of many scientific papers resulting from research
funded by the National Institutes of Health, the National Science Foundation, and
the American Cancer Society. He has also coauthored two genetics textbooks and
has written two articles on molecular biology in the National Geographic
Magazine. He has spent two years performing research in European laboratories
as an American Cancer Society Research Scholar, one year in Zurich, Switzerland,
and one year in Oxford, England.

iv


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B R I E F

C O N T E N T S

About the Author iv
Preface xiii
Acknowledgments xvii
Guide to Experimental Techniques in
Molecular Biology xix

PA R T V

PA R T I

Post-Transcriptional Events

Introduction

14 RNA Processing I: Splicing 394
15 RNA Processing II: Capping and Polyadenylation 436
16 Other RNA Processing Events and Post-Transcriptional
Control of Gene Expression 471

1 A Brief History 1
2 The Molecular Nature of Genes 12
3 An Introduction to Gene Function 30
PA R T I I

Methods in Molecular Biology
4 Molecular Cloning Methods 49
5 Molecular Tools for Studying Genes and Gene
Activity 75
PA R T I I I

Transcription in Bacteria

Translation
17 The Mechanism of Translation I: Initiation 522
18 The Mechanism of Translation II: Elongation
and Termination 560
19 Ribosomes and Transfer RNA 601
PA R T V I I

6 The Mechanism of Transcription in Bacteria 121
7 Operons: Fine Control of Bacterial
Transcription 167
8 Major Shifts in Bacterial Transcription 196
9 DNA–Protein Interactions in Bacteria 222
PA R T I V

Transcription in Eukaryotes
10 Eukaryotic RNA Polymerases and
Their Promoters 244

11 General Transcription Factors
in Eukaryotes 273
12 Transcription Activators in Eukaryotes
13 Chromatin Structure and Its Effects
on Transcription 355

PA R T V I

DNA Replication, Recombination,
and Transposition
20
21
22
23

DNA Replication, Damage, and Repair 636
DNA Replication II: Detailed Mechanism 677
Homologous Recombination 709
Transposition 732

PA R T V I I I

Genomes

314

24 Introduction to Genomics: DNA Sequencing on
a Genomic Scale 759
25 Genomics II: Functional Genomics, Proteomics,
and Bioinformatics 789

Glossary 827
Index 856
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O

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N

T

S

About the Author iv
Preface xiii
Acknowledgments xvii
Guide to Experimental Techniques
in Molecular Biology xix

CHAPTER 3

An Introduction to Gene Function 30
3.1

3.2
3.3

1

35

39

Replication 45
Mutations 45
45

2

Genetic Recombination and Mapping

4

Physical Evidence for Recombination

5

The Discovery of DNA

37

2

The Chromosome Theory of Inheritance

Molecular Genetics

31

40

Sickle Cell Disease

Mendel’s Laws of Inheritance

3
PA R T I I

Methods of Molecular Biology

5
5

CHAPTER 4

The Relationship Between Genes and Proteins

1.3

Protein Function

Translation

Transmission Genetics

Activities of Genes

31

Transcription

CHAPTER 1

1.2

Protein Structure

Discovery of Messenger RNA

Introduction

1.1

31

Overview of Gene Expression

PA R T I

A Brief History

Storing Information

6

7

The Three Domains of Life

Molecular Cloning Methods
4.1

9

Gene Cloning

50

The Role of Restriction Endonucleases
Vectors

49
50

53

Identifying a Specific Clone with a
Specific Probe 58

CHAPTER 2

cDNA Cloning

The Molecular Nature of Genes
2.1

The Nature of Genetic Material
Transformation in Bacteria

DNA Structure

2.3
2.4

13

The Polymerase Chain Reaction

62

62

Using Reverse Transcriptase PCR (RT-PCR)
in cDNA Cloning 64

19

Real-Time PCR

Genes Made of RNA 22
Physical Chemistry of Nucleic Acids
23

DNAs of Various Sizes and Shapes
vi

61

Box 4.1 Jurassic Park: More than a Fantasy?

15

19

A Variety of DNA Structures

Rapid Amplification of cDNA Ends
Standard PCR

18

Experimental Background
The Double Helix

4.2

13

The Chemical Nature of Polynucleotides

2.2

12

60

27

4.3
23

64

Methods of Expressing Cloned Genes
Expression Vectors

65

Other Eukaryotic Vectors

71

Using the Ti Plasmid to Transfer Genes
to Plants 71

65

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Contents

CHAPTER 5

Molecular Tools for Studying Genes
and Gene Activity 75
5.1

Molecular Separations
Gel Electrophoresis

76

80

Gel Filtration Chromatography

80

82

Autoradiography

82

Phosphorimaging

83

79

PA R T I I I

CHAPTER 6
84

The Mechanism of Transcription
in Bacteria 121

84

Using Nucleic Acid Hybridization

85

Southern Blots: Identifying Specific DNA Fragments 85
DNA Fingerprinting and DNA Typing

6.2

122

123

89

126

Sigma Stimulates Transcription Initiation

89

Reuse of s

129

Automated DNA Sequencing

91

High-Throughput Sequencing

93

Promoter Clearance

S1 Mapping

6.4

6.5

102

Nuclear Run-On Transcription

Reporter Gene Transcription

Rho-Dependent Termination

104

Assaying DNA–Protein Interactions

106

156
159

108

Operons: Fine Control of Bacterial
Transcription 167
7.1

The lac Operon

168

Negative Control of the lac Operon

109

Discovery of the Operon

109

The Mechanism of Repression
112

112

169

169

Repressor–Operator Interactions

DMS Footprinting and Other Footprinting
Methods 109

Assaying Protein–Protein Interactions

146

156

CHAPTER 7

108

Chromatin Immunoprecipitation (ChIP)

144

104

105

Measuring Protein Accumulation in Vivo

Termination of Transcription
Rho-Independent Termination

Measuring Transcription Rates in Vivo

DNase Footprinting

144

Structure of the Elongation Complex

100

Gel Mobility Shift

Elongation

Core Polymerase Functions in Elongation

Run-Off Transcription and G-Less
Cassette Transcription 103

Filter Binding

139

The Role of the a-Subunit in UP Element
Recognition 142

99

Primer Extension

132

134

Structure and Function of s

Protein Engineering with Cloned Genes:
Site-Directed Mutagenesis 97
Mapping and Quantifying Transcripts 99

127

128

The Stochastic s-Cycle Model

95

123

125

Transcription Initiation

Local DNA Melting at the Promoter

Northern Blots

5.9

Promoters

Promoter Structure

6.3

DNA Sequencing and Physical Mapping

Restriction Mapping

5.8

122

Binding of RNA Polymerase to Promoters

The Sanger Chain-Termination
Sequencing Method 90

5.7

RNA Polymerase Structure

Sigma (s) as a Specificity Factor

In Situ Hybridization: Locating Genes in
Chromosomes 88
Immunoblots (Western Blots)

6.1

86

Forensic Uses of DNA Fingerprinting and
DNA Typing 87

5.6

115

Transcription in Bacteria

Nonradioactive Tracers

5.5

115

115

Transgenic Mice

81

Liquid Scintillation Counting

5.4

114

5.11 Knockouts and Transgenics

Ion-Exchange Chromatography

Labeled Tracers

114

Functional SELEX
Knockout Mice

Affinity Chromatography

5.3

SELEX

76

Two-Dimensional Gel Electrophoresis

5.2

5.10 Finding RNA Sequences That Interact
with Other Molecules 114

173

174

Positive Control of the lac Operon
The Mechanism of CAP Action

177

178

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The ara Operon

182

9.4

The ara Operon Repression Loop

183

Evidence for the ara Operon Repression Loop

7.3

DNA-Binding Proteins: Action at
a Distance 237
The gal Operon

183

237

Autoregulation of araC 185

Duplicated l Operators

The trp Operon

Enhancers

186

237

238

Tryptophan’s Role in Negative Control of the
trp Operon 186
Control of the trp Operon by Attenuation
Defeating Attenuation

7.4

Riboswitches

187
PA R T I V

188

Transcription in Eukaryotes

190

CHAPTER 8

CHAPTER 10

Major Shifts in Bacterial
Transcription 196
8.1

Sigma Factor Switching

Phage Infection
Sporulation

Eukaryotic RNA Polymerases
and Their Promoters 244

197

10.1

197

199

Separation of the Three Nuclear Polymerases

Genes with Multiple Promoters 201
Other s Switches
Anti-s-Factors

8.2
8.3

The Roles of the Three RNA Polymerases

201

RNA Polymerase Subunit Structures

202

The RNA Polymerase Encoded in
Phage T7 202
Infection of E. coli by Phage l 203
Lytic Reproduction of Phage l
Establishing Lysogeny

204

211

10.2

Determining the Fate of a l Infection: Lysis or
Lysogeny 217
Lysogen Induction

218

DNA–Protein Interactions
in Bacteria 222

263

Class III Promoters

264

Enhancers and Silencers
Enhancers

Silencers

267

267
269

CHAPTER 11

11.1

Class II Factors

274

The Class II Preinitiation Complex

274

Structure and Function of TFIID

276

Probing Binding Specificity by SiteDirected Mutagenesis 223

Structure and Function of TFIIB

286

Structure and Function of TFIIH

288

Box 9.1 X-Ray Crystallography

The Mediator Complex and the
RNA Polymerase II Holoenzyme

295

The l Family of Repressors

223

224

High-Resolution Analysis of l Repressor–Operator
Interactions 229
High-Resolution Analysis of Phage 434
Repressor–Operator Interactions 232

The trp Repressor

Elongation Factors

11.2

234

The Role of Tryptophan

9.3

259

Class I Promoters

10.3

248

259

General Transcription Factors
in Eukaryotes 273

CHAPTER 9

9.2

Promoters

Class II Promoters

Autoregulation of the cI Gene During
Lysogeny 212

9.1

Multiple Forms of Eukaryotic

RNA Polymerase 245

234

General Considerations on
Protein–DNA Interactions 235

Class I Factors

296

299

The Core-Binding Factor

299

The UPE-Binding Factor

300

Structure and Function of SL1

11.3

Class III Factors

Hydrogen Bonding Capabilities of the Four
Different Base Pairs 235

TFIIIA

The Importance of Multimeric DNA-Binding Proteins 236

The Role of TBP

303

303

TFIIIB and C

304
307

301

245

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Contents

CHAPTER 12

Nucleosome Positioning

Transcription Activators
in Eukaryotes 314

Histone Acetylation

12.1

Categories of Activators
DNA-Binding Domains

Chromatin Remodeling

315

315

Homeodomains

Nucleosomes and Transcription
Elongation 387

315

Post-Transcriptional Events

318
319

CHAPTER 14

320

The bZIP and bHLH Domains

RNA Processing I: Splicing

321

Independence of the Domains
of Activators 323
Functions of Activators 324
Recruitment of TFIID

14.1

RNA Splicing

329

Box 12.1 Genomic Imprinting
Transcription Factories
Complex Enhancers
Enhanceosomes

334

Control of Splicing

343

14.3

344

Activator Acetylation

346

348

RNA Processing II: Capping and
Polyadenylation 436

Chromatin Structure and Its Effects
on Transcription 355
356

15.2

356

Capping

437

Cap Structure

437

Cap Synthesis

438

Functions of Caps

440

Polyadenylation

442

Poly(A)

357

442

Functions of Poly(A)

360

Higher-Order Chromatin Folding

13.2

430

CHAPTER 15

348

15.1

The 30-nm Fiber

427

Group II Introns

CHAPTER 13

Nucleosomes

427

347

Signal Transduction Pathways

Chromatin Structure

411

425

Self-Splicing RNAs
Group I Introns

Activator Sumoylation

Histones

402

Commitment, Splice Site Selection, and Alternative
Splicing 415

338

Regulation of Transcription Factors

399

401

Spliceosome Assembly and Function

337

398

The Mechanism of Splicing of Nuclear
mRNA Precursors 399

Spliceosomes

336

Activator Ubiquitylation

13.1

397

A Signal at the Branch

339

Coactivators

395

A Branched Intermediate

332

Architectural Transcription Factors
Insulators

14.2

328

Action at a Distance

395

Effect of Splicing on Gene Expression

328

394

396

Splicing Signals

325

Interaction Among Activators
Dimerization

Genes in Pieces

Evidence for Split Genes

324

Recruitment of the Holoenzyme

12.6

383

PA R T V

The Nuclear Receptors

12.5

376

Heterochromatin and Silencing

316

The GAL4 Protein

12.4

373

Structures of the DNA-Binding
Motifs of Activators 316
Zinc Fingers

12.3

372

Histone Deacetylation

Transcription-Activating Domains

12.2

367

362

Chromatin Structure and Gene
Activity 364
The Effects of Histones on Transcription
of Class II Genes 365

443

Basic Mechanism of Polyadenylation
Polyadenylation Signals

445

446

Cleavage and Polyadenylation of a Pre-mRNA
Poly(A) Polymerase

454

Turnover of Poly(A)

454

448

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15.3

Coordination of mRNA Processing Events

456

PA R T V I

Binding of the CTD of Rpb1 to mRNA-Processing
Proteins 457

Translation

Changes in Association of RNA-Processing Proteins
with the CTD Correlate with Changes in CTD
Phosphorylation 458

CHAPTER 17

A CTD Code?

460

Coupling Transcription Termination with mRNA
39-End Processing 461
Mechanism of Termination

The Mechanism of Translation I:
Initiation 522
17.1

462

tRNA Charging

Role of Polyadenylation in mRNA Transport

466

Other RNA Processing Events and
Post-Transcriptional Control of Gene
Expression 471
Ribosomal RNA Processing
Eukaryotic rRNA Processing
Bacterial rRNA Processing

16.2

17.3

472

475

537

545
545
548

475
CHAPTER 18

Forming Mature 39-Ends

476

The Mechanism of Translation II:
Elongation and Termination 560

Trans-Splicing 477
RNA Editing

477

18.1

479
479

18.2
482

Mechanism of RNAi

The Triplet Code

The (Almost) Universal Code

18.3

Processing Bodies

Other Small RNAs

517

569

Elongation Step 3: Translocation
G Proteins and Translation

511

514

570

18.4

Termination

580

583

584

Termination Codons
Release Factors

577

582

The Structures of EF-Tu and EF-G

507

584

Stop Codon Suppression

Degradation of mRNAs in P-Bodies
Relief of Repression in P-Bodies

569

Overview of Elongation

Elongation Step 2: Peptide Bond Formation

502

510

The Elongation Cycle

Elongation Step 1: Binding an Aminoacyl-tRNA to the
A Site of the Ribosome 573

Piwi-Interacting RNAs and Transposon Control 501
Post-Transcriptional Control of Gene
Expression: MicroRNAs 502

Translation Repression, mRNA Degradation,
and P-Bodies 510

567

A Three-Site Model of the Ribosome

Role of the RNAi Machinery in Heterochromatin
Formation and Gene Silencing 495

Stimulation of Translation by miRNAs

564

Unusual Base Pairs Between Codon and Anticodon 566

494

Silencing of Translation by miRNAs

562

563

563

Breaking the Code

484

489

Amplification of siRNA

No Gaps in the Code

484

Post-Transcriptional Control of Gene Expression:
RNA Interference 488

The Direction of Polypeptide Synthesis and
of mRNA Translation 561
The Genetic Code 562
Nonoverlapping Codons

Post-Transcriptional Control of Gene
Expression: mRNA Stability 483
Transferrin Receptor mRNA Stability

16.9

Control of Initiation

533

475

Casein mRNA Stability

16.7
16.8

533

Eukaryotic Translational Control

Editing by Nucleotide Deamination

16.6

Initiation in Eukaryotes

533

Forming Mature 59-Ends

Mechanism of Editing

16.5

531

Bacterial Translational Control

The Mechanism of Trans-Splicing

16.4

525

Formation of the 70S Initiation Complex

Eukaryotic Initiation Factors

472

Cutting Apart Polycistronic Precursors

16.3

Formation of the 30S Initiation Complex

The Scanning Model of Initiation

474

Transfer RNA Processing

523

Summary of Initiation in Bacteria

17.2

523

523

Dissociation of Ribosomes

CHAPTER 16

16.1

Initiation of Translation in Bacteria

586

586

Dealing with Aberrant Termination

588

Use of Stop Codons to Insert Unusual Amino Acids 593


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18.5

Posttranslation

593

Excision Repair

Folding Nascent Proteins

660

Double-Strand Break Repair in Eukaryotes

594

Release of Ribosomes from mRNA

Mismatch Repair

595

Failure of Mismatch Repair in Humans

CHAPTER 19

Ribosomes

CHAPTER 21

Ribosome Composition

DNA Replication II:
Detailed Mechanism

602

605

Fine Structure of the 30S Subunit

606

Fine Structure of the 50S Subunit

612

21.1

621

Transfer RNA

The Discovery of tRNA

tRNA Structure

678

Priming in Eukaryotes

21.2

623

Initiation

677

Priming in E. coli 678

Ribosome Structure and the Mechanism of Translation 616

19.2

668

601

602

Fine Structure of the 70S Ribosome

Polysomes

668

Coping with DNA Damage Without Repairing It

Ribosomes and Transfer RNA
19.1

665

667

Elongation

683

Speed of Replication

623

679
683

The Pol III Holoenzyme and Processivity
of Replication 683

623

Recognition of tRNAs by Aminoacyl-tRNA Synthetase:
The Second Genetic Code 626

21.3

Termination

694

Decatenation: Disentangling Daughter DNAs

Proofreading and Editing by Aminoacyl-tRNA
Synthetases 630

Termination in Eukaryotes

694

695

Box 21.1 Telomeres, the Hayflick Limit, and
Cancer 699
PA R T V I I

Telomere Structure and Telomere-Binding Proteins in
Lower Eukaryotes 702

DNA Replication, Recombination,
and Transposition

CHAPTER 22

CHAPTER 20

Homologous Recombination
22.1

DNA Replication, Damage,
and Repair 636
20.1

22.2

General Features of DNA Replication
Semiconservative Replication

Bidirectional Replication
Rolling Circle Replication

20.2

RuvC

642

22.3

645

646

721

722

Creation of Single-Stranded Ends at DSBs

650

22.4

651

Gene Conversion

728

651
CHAPTER 23

653

DNA Damage and Repair

719

Meiotic Recombination

The Double-Stranded DNA Break

Single-Strand DNA-Binding Proteins

20.3

717

The Mechanism of Meiotic Recombination:
Overview 721

649

Multiple Eukaryotic DNA Polymerases

Topoisomerases

715

RuvA and RuvB

Three DNA Polymerases in E. coli 646

Strand Separation

712

RecBCD

639

641

Enzymology of DNA Replication
Fidelity of Replication

The RecBCD Pathway for Homologous
Recombination 710
Experimental Support for the RecBCD
Pathway 712
RecA

637

At Least Semidiscontinuous Replication
Priming of DNA Synthesis

637

709

Transposition

656

Damage Caused by Alkylation of Bases
Damage Caused by Ultraviolet Radiation
Damage Caused by Gamma and X-Rays
Directly Undoing DNA Damage

659

657
658
658

23.1

732

Bacterial Transposons 733
Discovery of Bacterial Transposons

733

Insertion Sequences: The Simplest Bacterial
Transposons 733

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More Complex Transposons

734

Mechanisms of Transposition

23.2

23.3

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Eukaryotic Transposons

734

Sequencing Standards

737

24.3

Studying and Comparing Genomic Sequences 774

P Elements

Other Vertebrate Genomes

Rearrangement of Immunoglobulin Genes

Retrotransposons
Retroviruses

Personal Genomics

739

740

The Barcode of Life

779
779

782
784

743
CHAPTER 25

743

Genomics II: Functional Genomics,
Proteomics, and Bioinformatics 789

745

745

Retrotransposons

774

The Minimal Genome

742

Mechanism of V(D)J Recombination

749

25.1

799

Single-Nucleotide Polymorphisms:
Pharmacogenomics 810

CHAPTER 24

25.2

Introduction to Genomics: DNA
Sequencing on a Genomic Scale 759
Positional Cloning: An Introduction
to Genomics 760
Classical Tools of Positional Cloning

765

767

Vectors for Large-Scale Genome Projects
770

812

Protein Separations

Protein Analysis

769

812

813

Quantitative Proteomics

25.3

760

Techniques in Genomic Sequencing

Proteomics

Protein Interactions

Identifying the Gene Mutated in a Human Disease 762

The Clone-by-Clone Strategy

790

Genomic Functional Profiling

Genomes

The Human Genome Project

Functional Genomics: Gene Expression
on a Genomic Scale 790
Transcriptomics

PA R T V I I I

24.2

774

The Human Genome

The Recombinase

24.1

773

The First Examples of Transposable Elements:
Ds and Ac of Maize 737

Recombination Signals

23.4

Shotgun Sequencing

Bioinformatics

814

816

820

Finding Regulatory Motifs in Mammalian
Genomes 820
Using the Databases Yourself

Glossary 827
Index 856

822


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P

One of my most exciting educational experiences was my
introductory molecular biology course in graduate school.
My professor used no textbook, but assigned us readings
directly from the scientific literature. It was challenging,
but I found it immensely satisfying to meet the challenge
and understand, not only the conclusions, but how the
evidence supported those conclusions.

When I started teaching my own molecular biology
course, I adopted this same approach, but tried to reduce
the challenge to a level more appropriate for undergraduate students. I did this by narrowing the focus to the most
important experiments in each article, and explaining
those carefully in class. I used hand-drawn cartoons and
photocopies of the figures as illustrations.
This approach worked well, and the students enjoyed
it, but I really wanted a textbook that presented the concepts of molecular biology, along with experiments that
led to those concepts. I wanted clear explanations that
showed students the relationship between the experiments
and the concepts. So, I finally decided that the best way to
get such a book would be to write it myself. I had already
coauthored a successful introductory genetics text in
which I took an experimental approach—as much as possible with a book at that level. That gave me the courage
to try writing an entire book by myself and to treat the
subject as an adventure in discovery.

Organization
The book begins with a four-chapter sequence that should
be a review for most students. Chapter 1 is a brief history
of genetics. Chapter 2 discusses the structure and chemical
properties of DNA. Chapter 3 is an overview of gene expression, and Chapter 4 deals with the nuts and bolts of
gene cloning. All these are topics that the great majority
of molecular biology students have already learned in an
introductory genetics course. Still, students of molecular
biology need to have a grasp of these concepts and may
need to refresh their understanding of them. I do not deal
specifically with these chapters in class; instead, I suggest
students consult them if they need more work on these topics.
These chapters are written at a more basic level than the

rest of the book.
Chapter 5 describes a number of common techniques
used by molecular biologists. It would not have been possible to include all the techniques described in this book in
one chapter, so I tried to include the most common or, in a

R

E

F

A

C

E

few cases, valuable techniques that are not mentioned elsewhere in the book. When I teach this course, I do not present Chapter 5 as such. Instead, I refer students to it when
we first encounter a technique in a later chapter. I do it that
way to avoid boring my students with technique after technique. I also realize that the concepts behind some of these
techniques are rather sophisticated, and the students’ appreciation of them is much deeper after they’ve acquired
more experience in molecular biology.
Chapters 6–9 describe transcription in bacteria. Chapter 6 introduces the basic transcription apparatus, including promoters, terminators, and RNA polymerase, and
shows how transcripts are initiated, elongated, and terminated. Chapter 7 describes the control of transcription in
three different operons, then Chapter 8 shows how bacteria and their phages control transcription of many genes at
a time, often by providing alternative sigma factors. Chapter 9 discusses the interaction between bacterial DNAbinding proteins, mostly helix-turn-helix proteins, and
their DNA targets.
Chapters 10–13 present control of transcription in eukaryotes. Chapter 10 deals with the three eukaryotic RNA
polymerases and the promoters they recognize. Chapter 11
introduces the general transcription factors that collaborate with the three RNA polymerases and points out the

unifying theme of the TATA-box-binding protein, which
participates in transcription by all three polymerases.
Chapter 12 explains the functions of gene-specific transcription factors, or activators. This chapter also illustrates
the structures of several representative activators and
shows how they interact with their DNA targets. Chapter 13
describes the structure of eukaryotic chromatin and shows
how activators and silencers can interact with coactivators
and corepressors to modify histones, and thereby to activate
or repress transcription.
Chapters 14–16 introduce some of the posttranscriptional events that occur in eukaryotes. Chapter 14 deals
with RNA splicing. Chapter 15 describes capping and
polyadenylation, and Chapter 16 introduces a collection of
fascinating “other posttranscriptional events,” including
rRNA and tRNA processing, trans-splicing, and RNA editing. This chapter also discusses four kinds of posttranscriptional control of gene expression: (1) RNA interference;
(2) modulating mRNA stability (using the transferrin receptor
mRNA as the prime example); (3) control by microRNAs,
and (4) control of transposons in germ cells by Piwi-interacting
RNAs (piRNAs).
xiii


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Preface

Chapters 17–19 describe the translation process in both

bacteria and eukaryotes. Chapter 17 deals with initiation
of  translation, including the control of translation at the
initiation step. Chapter 18 shows how polypeptides are
elongated, with the emphasis on elongation in bacteria.
Chapter 19 provides details on the structure and function of
two of the key players in translation: ribosomes and tRNA.
Chapters 20–23 describe the mechanisms of DNA replication, recombination, and translocation. Chapter 20 introduces the basic mechanisms of DNA replication and
repair, and some of the proteins (including the DNA polymerases) involved in replication. Chapter 21 provides details of the initiation, elongation, and termination steps in
DNA replication in bacteria and eukaryotes. Chapters 22
and 23 describe DNA rearrangements that occur naturally
in cells. Chapter 22 discusses homologous recombination
and Chapter 23 deals with translocation.
Chapters 24 and 25 present concepts of genomics, proteomics, and bioinformatics. Chapter 24 begins with an oldfashioned positional cloning story involving the Huntington
disease gene and contrasts this lengthy and heroic quest
with the relative ease of performing positional cloning with
the human genome (and other genomes). Chapter 25 deals
with functional genomics (transcriptomics), proteomics,
and bioinformatics.

New to the Fifth Edition
The most obvious change in the fifth edition is the splitting
of old Chapter 24 (Genomics, Proteomics, and Bioinformatics) in two. This chapter was already the longest in the book,
and the field it represents is growing explosively, so a split
was inevitable. The new Chapter 24 deals with classical genomics: the sequencing and comparison of genomes. New
material in Chapter 24 includes an analysis of the similarity
between the human and chimpanzee genomes, and a look at
the even closer similarity between the human and Neanderthal genomes, including recent evidence for interbreeding
between humans and Neanderthals. It also includes an update on the new field of synthetic biology, made possible by
genomic work on microorganisms, and contains a report of
the recent success by Craig Venter and colleagues in creating

a living Mycoplasma cell with a synthetic genome.
Chapter 25 deals with fields allied with Genomics:
Functional Genomics, Proteomics, and Bioinformatics.
New material in Chapter 25 includes new applications of
the ChIP-chip and ChIP-seq techniques—the latter using
next-generation DNA sequencing; collision-induced dissociation mass spectrometry, which can be used to sequence
proteins; and the use of isotope-coded affinity tags (ICATs)
and stable isotope labeling by amino acids (SILAC) to
make mass spectrometry (MS) quantitative. Quantitative
MS in turn enables comparative proteomics, in which the
concentrations of large numbers of proteins can be compared between species.

All but the introductory chapters of this fifth edition
have been updated. Here are a few highlights:
• Chapter 5: Introduces high-throughput (next
generation) DNA sequencing techniques. These have
revolutionized the field of genomics. Chromatin
immunoprecipitation (ChIP) and the yeast two-hybrid
assay have been moved to Chapter 5, in light of their
broad applicabilities. A treatment of the energies of
the b-electrons from 3H, 14C, 35S, and 32P has been
added, and the fluorography technique, which captures information from the lower-energy emissions,
is discussed.
• Chapter 6: Adds a discussion of FRET-ALEX (FRET
with alternating laser excitation), along with a description of how this technique has been used to
support (1) the stochastic release model of the
s-cycle and (2) the scrunching hypothesis to explain
abortive transcription. This chapter also updates
the structure of the bacterial elongation complex,
including a discussion of a two-state model for

nucleotide addition.
• Chapter 7: Introduces the riboswitch in the mRNA
from the glmS gene of B. subtilis, in which the end
product of the gene turns expression of the gene off
by stimulating the mRNA to destroy itself. This
chapter also introduces a hammerhead ribozyme as
a possible mammalian riboswitch that may operate
by a similar mechanism.
• Chapter 8: Introduces the concepts of anti-s-factors
and anti-anti-s-factors as controllers of transcription
during sporulation in B. subtilis.
• Chapter 9: Emphasizes the dynamic nature of protein structure, and points out that a given crystal
structure represents just one of a range of different
possible protein conformations.
• Chapter 10: Presents a new study by Roger Kornberg’s group that identifies the RNA polymerase II
trigger loop as a key determinant in transcription
specificity, along with a discussion of how the
enzyme distinguishes between ribonuncleotides and
deoxyribonucleotides. This chapter also introduces
the concepts of core promoter and proximal promoter, where the core promoter contains any combination of TFIIB recognition element, TATA box,
initiator, downstream promoter element, downstream core element, and motif ten element, and
the proximal promoter contains upstream
promoter elements.
• Chapter 11: Introduces the concept of core TAFs—
those associated with class II preinitiation complexes
from a wide variety of eukaryotes, and introduces
the new nomenclature (TAF1–TAF13), which
replaces the old, confusing nomenclature that was

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Preface

based on molecular masses (e.g., TAFII250). This
chapter also describes an experiment that shows the
importance of TFIIB in setting the start site of transcription. It also shows that a similar mechanism
applies in the archaea, which use a TFIIB homolog
known as transcription factor B.
• Chapter 12: Introduces the technique of chromosome
conformation capture (3C) and shows how it can be
used to detect DNA looping between an enhancer
and a promoter. This chapter also introduces the concept of imprinting during gametogenesis, and explains
the role of methylation in imprinting, particularly
methylation of the imprinting control region of the
mouse Igf2/H19 locus. It also introduces the concept
of transcription factories, where transcription of multiple genes occurs. Finally, this chapter refines and
updates the concept of the enhanceosome.
• Chapter 13: Presents a new table showing all the
ways histones can be modified in vivo; brings back
the solenoid, alongside the two-start helix, as a
candidate for the 30-nm fiber structure; and presents
evidence that chromatin adopts one or the other
structure, depending on its nucleosome repeat
length. This chapter also introduces the concept of
specific histone methylations as markers for transcription initiation and elongation, and shows how
this information can be used to infer that RNA polymerase II is poised between initiation and elongation
on many human protein-encoding genes. It also

emphasizes the importance of histone modifications
in affecting not only histone–DNA interactions, but
also nucleosome–nucleosome interactions and
recruitment of histone-modifying and chromatinremodeling proteins. Finally, this chapter shows how
PARP1 (poly[ADP-ribose] polymerase-1) can
facilitate nucleosome loss from chromatin by
poly(ADP-ribosyl)ating itself.
• Chapter 14: Introduces the exon junction complex
(EJC), which is added to mRNAs during splicing in
the nucleus, and shows how the EJC can stimulate
transcription by facilitating the association of
mRNAs with ribosomes. This chapter also introduces exon and intron definition modes of splicing
and shows how they can be distinguished experimentally. This test has revealed that higher eukaryotes primarily use exon definition and lower
eukaryotes primarily use intron definition.
• Chapter 15: Demonstrates that a subunit of CPSF
(CPSF-73) is responsible for cutting a pre-mRNA at
a polyadenylation signal. It also shows that serine 7,
in addition to serines 2 and 5 in the repeating
heptad in the CTD of the largest RNA polymerase
subunit, can be phosphorylated, and shows that this
serine 7 phosphorylation controls the expression of

xv

certain genes (e.g., the U2 snRNA gene) by controlling the 39-end processing of their mRNAs.
• Chapter 16: Identifies a single enzyme, tRNA 39 processing endoribonuclease, as the agent that cleaves
excess nucleotides from the 39-end of a eukaryotic
tRNA precursor; points out the overwhelming prevalence of trans-splicing in C. elegans; presents a new
model for removal of the passenger strand of a
double-stranded siRNA—cleavage of the passenger

strand by Ago2; introduces Piwi-interacting RNAs
(piRNAs) and presents the ping-pong model by
which they are assumed to amplify themselves and
inactivate transposons in germ cells; introduces
plant RNA polymerases IV and V, and describes
their roles in gene silencing. This chapter also greatly
expands the coverage of miRNAs, and points out
that hundreds of miRNAs control thousands of
plant and animal genes, and that mutations in
miRNA genes typically have very deleterious effects.
Chapter 16 also updates the biogenesis of miRNAs,
introducing two pathways to miRNA production:
the Drosha and mirtron pathways. Finally, this
chapter introduces P-bodies, which are involved in
mRNA decay and translational repression.
• Chapter 17: Updates the section on eukaryotic
viral internal ribosome entry sequences (IRESs).
Some viruses cleave eIF4G, leaving a remnant
called p100. Poliovirus IRESs bind to p100 and
thereby gain access to ribosomes, but hepatitis C
virus IRESs bind directly to eIF3, while hepatitis A
virus IRESs bind even more directly to ribosomes.
This chapter also refines the model describing how
the cleavage of eIF4G affects mammalian host
mRNA translation. Different cell types respond
differently to this cleavage. Finally, this chapter
introduces the concept of the pioneer round of
translation, and points out that different initiation
factors are used in the pioneer round than in all
subsequent rounds.

• Chapter 18: Introduces the concept of superwobble,
which holds that a single tRNA with a U in its wobble position can recognize codons ending in any of
the four bases, and presents evidence that superwobble works. This chapter also introduces the hybrid P/I
state as the initial ribosomal binding state for fMettRNA Met
f . In this state, the anticodon is in the P site,
but the fMet and acceptor stem are in an “initiator”
site between the P site and the E site. This chapter
also describes no-go decay, which degrades mRNA
containing a stalled ribosome, and introduces the
concept of codon bias to explain inefficiency of
translation. Finally, this chapter explains how the
slowing of translation by rare codons can influence
protein folding both negatively and positively.


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Preface

• Chapter 19: Includes a new section based on recent
crystal structures of the ribosome in complex with
various elongation factors. One of these structures
involves aminoacyl-tRNA and EF-Tu, and has
shown that the tRNA is bent by about 30 degrees in
forming an A/T complex. This bend is important in
fidelity of translation, and also facilitates the GTP

hydrolysis that permits EF-Tu to leave the ribosome.
Another crystal structure involves EF-G–GDP and
shows the ribosome in the post-translocation E/E,
P/P state, as opposed to the spontaneously achieved
pre-translocation P/E, A/P hybrid state. This chapter
also provides links to two excellent new movies describing the elongation process and an overview of
translation initiation, elongation, and termination.
Finally, this chapter describes crystal structures that
illustrate the functions of two critical parts of RF1
and RF2 in stop codon recognition and cleavage of
polypeptides from their tRNAs.
• Chapter 20: Introduces the controversial proposal,
with evidence, that DNA replication in E. coli is discontinuous on both strands. This chapter also introduces ACL1, a chromatin remodeler recruited via its
macrodomain to sites of double-strand breaks by
poly(ADP-ribose) formed at these sites by poly(ADPribose) polymerase 1 (PARP-1).
• Chapter 21: Presents a co-crystal structure of a b dimer bound to a primed DNA template, showing that
the b clamp really does encircle the DNA, but that
the DNA runs through the circle at an angle of
20 degrees with respect to the horizontal. This chapter
also includes a corrected and updated Figure 21.17
(model of the polIII* subassembly) to show a single
g-subunit and the two t-subunits joined to the core
polymerases through their flexible C-terminal domains. This section also clarifies that the g- and
t-subunits are products of the same gene, but the
former lacks the C-terminal domain of the latter.
This chapter also introduces the complex of telomerebinding proteins known as shelterin, and focuses on
the six shelterin proteins of mammals and their roles
in protecting telomeres, and in preventing inappropriate repair and cell cycle arrest in response to
normal chromosome ends.
• Chapter 22: Adds a new figure (Figure 22.3) to

show how different nicking patterns to resolve the
Holliday junction in the RecBCD pathway lead to
different recombination products (crossover or
noncrossover recombinants).
• Chapter 23: Reports that piRNAs targeting P element
transposons are likely to be the transposition
suppressors in the P-M system. Similarly, piRNAs
appear to play the suppressor role in the I-R transposon system.

Supplements
For the Student www.mhhe.com/weaver5e
The text website for Molecular Biology is a great place to
review chapter material and to enhance your study routine.
Here you will have access to:
• digital image files
• questions
• animation quizzes
• web links.
• PowerPoint lecture outlines
• answers to end-of-chapter
For the Instructor www.mhhe.com/weaver5e
The Molecular Biology website offers a wealth of teaching
and learning aids for instructors and students. Instructors
will appreciate:
• Test bank questions and software
options with EZ Test Online, desktop
version or Word docs.
• Answers to end-of-chapter questions
• Lecture outline PowerPoint files
• Image PowerPoint files

• McGraw-Hill Presentation Center
McGraw-Hill Presentation Center
Build instructional materials
wherever, whenever, and however you want! Presentation
Center is an online digital library containing assets such as
photos, artwork, PowerPoints, animations, and other media
types that can be used to create customized lectures, visually
enhanced tests and quizzes, compelling course websites, or
attractive printed support materials.

Options
You’re in charge of your course, so
why not be in control of the content of
your textbook? At McGraw-Hill Custom Publishing, we can help you create the ideal text—the
one you’ve always imagined. Quickly. Easily. With more
than 20 years of experience in custom publishing, we’re
experts. But at McGraw Hill, we’re also innovators,
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not only about saving trees; it’s also about saving money.
Available for a greatly reduced price, McGraw-Hill eBooks
are an eco-friendly and cost-savings alternative to the
traditional print textbook. So, you do some good for the
environment . . . and you do some good for your wallet.
Visit www.mhhe.com/ebooks for details.

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A C K N O W L E D G M E N T S

In writing this book, I have been aided immeasurably by the advice of many editors and reviewers. They have contributed
greatly to the accuracy and readability of the book, but they cannot be held accountable for any remaining errors or
ambiguities. For those, I take full responsibility. I would like to thank the following people for their help.
Fifth Edition Reviewers
Aimee Bernard
University of Colorado–
Denver
Brian Freeman
University of Illinois,
Urbana–Champaign
Dennis Bogyo
Valdosta State University
Donna Hazelwood
Dakota State University
Margaret Ritchey
Centre College
Nemat Kayhani
University of Florida
Nicole BourniasBardiabasis
California State University,
San Bernardino
Ruhul Kuddus
Utah Valley University
Tao Weitao

University of Texas at
San Antonio
Fourth Edition Reviewers
Dr. David Asch
Youngstown State University
Christine E. Bezotte
Elmira College
Mark Bolyard
Southern Illinois University,
Edwardsville
Diane Caporale
University of Wisconsin,
Stevens Point
Jianguo Chen
Claflin University

Chi-Lien Cheng
Department of Biological
Sciences, University of Iowa
Mary Ellard-Ivey
Pacific Lutheran University
Olukemi Fadayomi
Ferris State University, Big
Rapids, Michigan
Charles Giardina
University of Connecticut
Eli V. Hestermann
Furman University
Dr. Dorothy Hutter
Monmouth University

Cheryl Ingram-Smith
Clemson University
Dr. Cynthia Keler
Delaware Valley College
Jack Kennell
Saint Louis University
Charles H. Mallery
College of Arts and Sciences,
University of Miami
Jon L. Milhon
Azusa Pacific University
Hao Nguyen
California State University,
Sacramento
Thomas Peterson
Iowa State University
Ed Stellwag
East Carolina University
Katherine M. Walstrom
New College of Florida
Cornelius A. Watson
Roosevelt University
Fadi Zaher
Gateway Technical College

Third Edition Reviewers
David Asch
Youngstown State
University
Gerard Barcak

University of Maryland
School of Medicine
Bonnie Baxter
Hobart & William Smith
Colleges
André Bédard
McMaster University
Felix Breden
Simon Fraser University
Laura Bull
UCSF Liver Center
Laboratory
James Ellis
Developmental Biology
Program, Hospital for Sick
Children, Toronto, Ontario
Robert Helling
The University of
Michigan
David Hinkle
University of Rochester
Robert Leamnson
University of Massachusetts
at Dartmouth
David Mullin
Tulane University
Marie Pizzorno
Bucknell University
Michael Reagan
College of St. Benedict/

St. John’s University
Rodney Scott
Wheaton College

Second Edition Reviewers
Mark Bolyard
Southern Illinois University
M. Suzanne Bradshaw
University of Cincinnati
Anne Britt
University of California, Davis
Robert Brunner
University of California,
Berkeley
Caroline J. Decker
Washington State University
Jeffery DeJong
University of Texas, Dallas
Stephen J. D’Surney
University of Mississippi
John S. Graham
Bowling Green State
University
Ann Grens
Indiana University
Ulla M. Hansen
Boston University
Laszlo Hanzely
Northern Illinois University
Robert B. Helling

University of Michigan
Martinez J. Hewlett
University of Arizona
David C. Hinkle
University of Rochester
Barbara C. Hoopes
Colgate University
Richard B. Imberski
University of Maryland
Cheryl Ingram-Smith
Pennsylvania State
University
xvii


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Acknowledgments

Alan Kelly
University of Oregon
Robert N. Leamnson
University of Massachusetts,
Dartmouth
Karen A. Malatesta
Princeton University

Robert P. Metzger
San Diego State University
David A. Mullin
Tulane University
Brian K. Murray
Brigham Young University
Michael A. Palladino
Monmouth University
James G. Patton
Vanderbilt University
Martha Peterson
University of Kentucky
Marie Pizzorno
Bucknell University
Florence Schmieg
University of Delaware
Zhaomin Yang
Auburn University
First Edition Reviewers
Kevin L. Anderson
Mississippi State University
Rodney P. Anderson
Ohio Northern University

Prakash H. Bhuta
Eastern Washington
University
Dennis Bogyo
Valdosta State University
Richard Crawford

Trinity College
Christopher A. Cullis
Case Western Reserve
University
Beth De Stasio
Lawrence University
R. Paul Evans
Brigham Young University
Edward R. Fliss
Missouri Baptist College
Michael A. Goldman
San Francisco State
University
Robert Gregerson
Lyon College
Eileen Gregory
Rollins College
Barbara A. Hamkalo
University of California,
Irvine
Mark L. Hammond
Campbell University
Terry L. Helser
State University of New
York, Oneonta

Carolyn Herman
Southwestern College
Andrew S. Hopkins
Alverno College

Carolyn Jones
Vincennes University
Teh-Hui Kao
Pennsylvania State
University
Mary Evelyn B. Kelley
Wayne State University
Harry van Keulen
Cleveland State University
Leo Kretzner
University of South Dakota
Charles J. Kunert
Concordia University
Robert N. Leamnson
University of Massachusetts,
Dartmouth
James D. Liberatos
Louisiana Tech University
Cran Lucas
Louisiana State University
James J. McGivern
Gannon University
James E. Miller
Delaware Valley College
Robert V. Miller
Oklahoma State
University

George S. Mourad
Indiana University-Purdue

University
David A. Mullin
Tulane University
James R. Pierce
Texas A&M University,
Kingsville
Joel B. Piperberg
Millersville University
John E. Rebers
Northern Michigan
University
Florence Schmieg
University of Delaware
Brian R. Shmaefsky
Kingwood College
Paul Keith Small
Eureka College
David J. Stanton
Saginaw Valley State
University
Francis X. Steiner
Hillsdale College
Amy Cheng Vollmer
Swarthmore College
Dan Weeks
University of Iowa
David B. Wing
New Mexico Institute of
Mining & Technology

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GUIDE TO EXPERIMENTAL TECHNIQUES
IN MOLECULAR BIOLOGY

Technique
Activity gel assay
Affinity chromatography
Affinity labeling
Allele-specific RNAi
Autoradiography
Baculovirus expression vectors
Cap analysis of gene expression (CAGE)
cDNA cloning
ChIP-chip analysis
ChIP-seq analysis
Chromatin immunoprecipitation
(ChIP)
Chromosome conformation capture (3C)
Colony hybridization
CsCl gradient ultracentrifugation
Designing a probe by protein
microsequencing
Detecting DNA bending by
electrophoresis
DMS footprinting
DNA fingerprinting

DNA helicase assay
DNA microarrays
DNA microchips
DNA sequencing (automated)
DNA Sequencing (next generation,
high throughput)
DNA sequencing (Sanger chain
termination)
DNA typing
DNase footprinting
Dot blotting
End-filling
Epitope tagging
Exon trapping
Expressed sequence tags (ESTs)
Expression vectors
Far Western blotting
Filter-binding assay (DNA–protein
interaction)

Chapter

Page

Technique

Chapter

13
4

6
18
5
4
25
4
25
25

372
68
145
592
82
70
796
60
803
804

5
12
4
20

112
331
61
637

18

588

7
5
5
20
25
25
5

181
109
86
651
790
790
91

5

93

5
5
5
5
5
10

24
24
4
15

90
87
109
105
101
249
761
773
65
457

5

108

Fingerprinting (protein)
3
Fluorescence in situ hybridization (FISH)
5
Fluorescence resonance energy transfer (FRET) 6
Fluorography
5
FRET-ALEX
6
Footprinting (protein)

21
Functional SELEX
5
Gel electrophoresis (DNA)
5
Gel electrophoresis (protein)
5
Gel filtration chromatography
5
Gel mobility shift assay
5
Gene cloning with BACs
24
Gene cloning with cosmid vectors
4
Gene cloning with l phage vectors
4
Gene cloning with M13 phage vectors
4
Gene cloning with phagemid vectors
4
Gene cloning with plant vectors
4
Gene cloning with plasmid vectors
4
Gene cloning with YACs
24
G-less cassette transcription
5
Hybridization

2
Hydroxyl radical probing
18
Immunoblotting (Western blotting)
5
Immunoprecipitation
5
In situ expression analysis
25
In situ hybridization
5
Ion-exchange chromatography
5
Isoelectric focusing
5
Isotope coded affinity tags (ICAT)
25
Knockout mice
5
Linker scanning mutagenesis
10
Liquid scintillation counting
5
Mass spectrometry
25
Microsatellites
24
Nick translation
4
Northern blotting

5
Oligonucleotide-directed RNA
degradation
14
Oligonucleotide probe design
4

Page
46
88
129
83
135
691
114
76
78
80
109
769
57
55
57
58
71
53
769
104
26
596

89
106
808
88
80
79
814
115
261
84
813
771
60
99
407
59
xix


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Guide to Experimental Techniques in Molecular Biology

Technique
Phage display
Phosphorimaging

Plaque hybridization
Polymerase chain reaction (PCR)
Positional cloning
Primer extension
Protein fingerprinting
Protein footprinting
Protein sequencing by mass spectrometry
Pulse-chase labeling
Pulsed-field gel electrophoresis
(PFGE)
Radiation hybrid mapping
Rapid amplification of cDNA
ends (RACE)
Real-time PCR
Reporter gene transcription assay
Restriction fragment length
polymorphisms (RFLPs)
Restriction mapping
Reverse transcriptase PCR
(RT-PCR)
R-looping
RNA helicase assay
RNA interference (RNAi)
RNA–RNA cross-linking (with
4-thioU)
RNase mapping (RNase
protection assay)

Chapter

Page

25
5
4
4
24
5
3
21
25
16

818
83
55
62
760
102
46
691
813
484

5
24

78
772

4
4
5

61
64
105

24
5

760
95

4
14
17
16

64
395
540
488

14

404

5

102

Technique
Run-off transcription
Run-on transcription
S1 mapping
SDS-PAGE (protein)
SELEX (systematic evolution of
ligands by exponential enrichment)
Sequence-tagged sites (STSs)
Serial analysis of gene
expression (SAGE)
Shotgun sequencing
Single molecule DNA nanomanipulation
Single molecule force spectroscopy
Site-directed mutagenesis
Southern blotting
Stopped-flow kinetic assay
Synthetic lethal screen
Toeprinting
Topoisomerase assay
Transformation
Transgenic mice
Two-dimensional gel
electrophoresis
Ultracentrifugation
Variable number tandem
repeats (VNTRs)
X-ray crystallography
Yeast two-hybrid assay

Yeast two-hybrid screen

Chapter

Page

5
5
5
5

103
104
100
78

5
24

114
771

25
24
6
13
5
5
18
14

17
20
2
5

794
773
136
362
97
85
581
418
542
654
13
115

5
2

79
13–14

24
9
5
5

770

224
113
113


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C

H

A

P T

E

R

1

A Brief History

W
Garden pea flowers. Flower color (purple or white) was one of the
traits Mendel studied in his classic examination of inheritance in
the pea plant. © Shape‘n’colour/Alamy, RF.

hat is molecular biology? The
term has more than one definition. Some
define it very broadly as the attempt to
understand biological phenomena in
molecular terms. But this definition makes
molecular biology difficult to distinguish
from another well-known discipline, biochemistry. Another definition is more
restrictive and therefore more useful: the
study of gene structure and function at
the molecular level. This attempt to explain
genes and their activities in molecular
terms is the subject matter of this book.
Molecular biology grew out of the disciplines of genetics and biochemistry. In
this chapter we will review the major early
developments in the history of this hybrid
discipline, beginning with the earliest
genetic experiments performed by Gregor
Mendel in the mid-nineteenth century.


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Chapter 1 / A Brief History

In Chapters 2 and 3 we will add more substance to this
brief outline. By definition, the early work on genes cannot be considered molecular biology, or even molecular

genetics, because early geneticists did not know the
molecular nature of genes. Instead, we call it transmission
genetics because it deals with the transmission of traits
from parental organisms to their offspring. In fact, the
chemical composition of genes was not known until 1944.
At that point, it became possible to study genes as molecules, and the discipline of molecular biology began.

1.1

Transmission Genetics

In 1865, Gregor Mendel (Figure 1.1) published his findings
on the inheritance of seven different traits in the garden
pea. Before Mendel’s research, scientists thought inheritance occurred through a blending of each trait of the
parents in the offspring. Mendel concluded instead that
inheritance is particulate. That is, each parent contributes
particles, or genetic units, to the offspring. We now call
these particles genes. Furthermore, by carefully counting
the number of progeny plants having a given phenotype, or
observable characteristic (e.g., yellow seeds, white flowers),
Mendel was able to make some important generalizations.
The word phenotype, by the way, comes from the same
Greek root as phenomenon, meaning appearance. Thus, a
tall pea plant exhibits the tall phenotype, or appearance.
Phenotype can also refer to the whole set of observable
characteristics of an organism.

Mendel’s Laws of Inheritance
Mendel saw that a gene can exist in different forms called
alleles. For example, the pea can have either yellow or

green seeds. One allele of the gene for seed color gives rise
to yellow seeds, the other to green. Moreover, one allele can
be dominant over the other, recessive, allele. Mendel demonstrated that the allele for yellow seeds was dominant
when he mated a green-seeded pea with a yellow-seeded
pea. All of the progeny in the first filial generation (F1) had
yellow seeds. However, when these F1 yellow peas were allowed to self-fertilize, some green-seeded peas reappeared.
The ratio of yellow to green seeds in the second filial generation (F2) was very close to 3:1.
The term filial comes from the Latin: filius, meaning
son; filia, meaning daughter. Therefore, the first filial generation (F1) contains the offspring (sons and daughters) of
the original parents. The second filial generation (F2) is the
offspring of the F1 individuals.
Mendel concluded that the allele for green seeds must
have been preserved in the F1 generation, even though it
did not affect the seed color of those peas. His explanation

Figure 1.1 Gregor Mendel. (Source: © Pixtal/age Fotostock RF.)

was that each parent plant carried two copies of the gene;
that is, the parents were diploid, at least for the characteristics he was studying. According to this concept,
homozygotes have two copies of the same allele, either two
alleles for yellow seeds or two alleles for green seeds.
Heterozygotes have one copy of each allele. The two parents in the first mating were homozygotes; the resulting F1
peas were all heterozygotes. Further, Mendel reasoned that
sex cells contain only one copy of the gene; that is, they
are haploid. Homozygotes can therefore produce sex cells,
or gametes, that have only one allele, but heterozygotes can
produce gametes having either allele.
This is what happened in the matings of yellow with
green peas: The yellow parent contributed a gamete with a
gene for yellow seeds; the green parent, a gamete with

a gene for green seeds. Therefore, all the F1 peas got one
allele for yellow seeds and one allele for green seeds. They
had not lost the allele for green seeds at all, but because
yellow is dominant, all the seeds were yellow. However,
when these heterozygous peas were self-fertilized, they produced gametes containing alleles for yellow and green color
in equal numbers, and this allowed the green phenotype to
reappear.
Here is how that happened. Assume that we have two
sacks, each containing equal numbers of green and yellow
marbles. If we take one marble at a time out of one sack
and pair it with a marble from the other sack, we will
wind up with the following results: one-quarter of the
pairs will be yellow/yellow; one-quarter will be green/green;
and the remaining one-half will be yellow/green. The
alleles for yellow and green peas work the same way.