2. Jones & Bartlett Learn1ng T1tles 1n B1olog1cal Sc1ence
AIDS: Science and Society, Seventh Edition
Hung Fan, Ross F. Conner, & Luis P. Villarreal
AIDS: The Biological Basis, Fifth Edition
Benjamin S. Weeks & I. Edward Alcamo
A/coma's Fundamentals of Microbiology, Ninth Edition
Jeffrey C. Pommerville
A/como's Fundamentals of Microbiology, Body Systems Edition, Second Edition
Jeffrey C. Pommerville
A/como's Microbes and Society, Third Edition
Benjamin S. Weeks
Aquatic Entomology
W. Patrick McCafferty & Arwin V. Provonsha
Bioethics: An Introduction to the History, Methods, and Practice, Third Edition
Nancy S. Jecker, Albert R. Jonsen, & Robert A. Pearlman
Bioimaging: Current Concepts in Light and Electron Microscopy
Douglas E. Chandler & Robert W. Roberson
Biomedical Graduate School: A Planning Guide to the Admissions Process
David J. McKean & Ted R. Johnson
Biomedical Informatics: A Data Use(s Guide
Jules J. Berman
Botany: An Introduction to Plant Biology, Fifth Edition
James D. Mauseth
Botany: A Lab Manual
Stacy Pfluger
Case Studiesfor Understanding the Human Body, Second Edition
Stanton Braude, Deena Goran, & Alexander Miceli
Clinical Information Systems: Overcoming Adverse Consequences
Dean F. Sittig & Joan S. Ash
Defending Evolution: A Guide to the Evolution/Creation Controversy
Brian J. Alters
The Ecology of Agroecosystems
John H. Vandermeer
Electron Microscopy, Second Edition
John J. Bozzola & Lonnie D. Russell
Encounters in Microbiology, Volume 1, Second Edition
Jeffrey C. Pommerville
Encounters in Microbiology, Volume 2
Jeffrey C. Pommerville
Essential Genetics: A Genomics Perspective, Sixth Edition
Daniel L. Hartl
Essentials of Molecular Biology, Fourth Edition
George M. Malacinski
Evolution: Principles and Processes
Brian K. Hall
Exploring Bioinformatics: A Project-Based Approach
Caroline St. Clair & Jonathan E. Visick
Exploring the Way Life Works: The Science of Biology
Mahlon Hoagland, Bert Dodson, & Judy Hauck
Fundamentals of Microbiology, Tenth Edition
Jeffrey C. Pommerville
Genetics: Analysis of Genes and Genomes, Eighth Edition
Daniel L. Hartl & Maryellen Ruvolo
Genetics of Populations, Fourth Edition
Philip W. Hedrick
Guide to Infectious Diseases by Body System, Second Edition
Jeffrey C. Pommerville
Human Biology, Seventh Edition
Daniel D. Chiras
Human Biology Laboratory Manual
Charles Welsh
Human Embryonic Stem Cells, Second Edition
Ann A. Kiessling & Scott C. Anderson
Introduction to the Biology of Marine Life, Tenth Edition
John F. Morrissey & James L. Sumich
Laboratory and Ae/d Investigations in Marine Life, Tenth Edition
Gordon H. Dudley, James L. Sumich, & Virginia L. Cass-Dudley
Laboratory Fundamentals of Microbiology, Ninth Edition
Jeffrey C. Pommerville
Laboratory Investigations in Molecular Biology
Steven A. Williams, Barton E. Slatko, & John R. McCarrey
Laboratory Textbook of Anatomy and Physiology: Cat Version, Ninth Edition
Anne B. Donnersberger
Lewin's CELLS, Second Edition
Lynne Cassimeris, Vishwanath R. Lingappa, & George Plopper
Lewin's Essential GENES, Third Edition
Jocelyn E. Krebs, Elliott S. Goldstein, & Stephen T. Kilpatrick
Mamma/ogy, Fifth Edition
Terry A. Vaughan, James M. Ryan, & Nicholas J. Czaplewski
The Microbial Challenge: Science, Disease, and Public Health, Third Edition
Robert I. Krasner
Microbial Genetics, Second Edition
Stanley R. Maloy, John E. Cronan, Jr., & David Freifelder
Microbiology Pearls of Wisdom, Second Edition
S. James Booth
Molecular Biology: Genes to Proteins, Fourth Edition
Burton E. Tropp
Neoplasms: Principles of Development and Diversity
Jules J. Berman
Perl Programmingfor Medicine and Bio/ogy
Jules J. Berman
Plant Biochemistry
Florence K. Gleason with Raymond Chollet
Plant Cell Biology
Brian E. S. Gunning & Martin W. Steer
Plants, Genes, and Crop Biotechnology, Second Edition
Maarten J. Chrispeels & David E. Sadava
Plants & People
James D. Mauseth
Plant Structure: A Color Guide, Second Edition
Bryan G. Bowes & James D. Mauseth
Precancer: The Beginning and the End of Cancer
Jules J. Berman
Principles of Cell Biology
George Plopper
Principles of Molecular Biology
Burton E. Tropp
Principles of Modern Microbiology
Mark Wheelis
Protein Microarrays
Mark Schena, ed.
Python for Bioinformatics
Jason Kinser
R for Medicine and Biology
Paul D. Lewis
Restoration Ecology
Sigurdur Greipsson
Ruby Programmingfor Medicine and Biology
Jules J. Berman
Strickberge(s Evolution, Fifth Edition
Brian K. Hall
Symbolic Systems Biology: Theory and Methods
M. Sriram Iyengar
Tropical Forests
Bernard A. Marcus
20th Century Microbe Hunters
Robert I. Krasner
Understanding Viruses, Second Edition
Teri Shors
3. '
JOCELYN E. KREBS
UNIVERSITY OF ALASKA, ANCHORAGE
ELLIOTT S. GOLDSTEIN
ARIZONA STATE UNIVERSITY
STEPHEN T. KILPATRICK
UNIVERSITY OF PITTSBURGH AT JOHNSTOWN
JONES & BARTLETI
LEARNING
5. Dedication
To Benjamin Lewin, for setting the bar high.
To my mother, Ellen Baker, for raising me with a love of science; to the memory of
my stepfather, Barry Kiefer, for convincing me science would stay fun; to my partner, Susannah Morgan,
for always pretending my biology jokes are funny; and to my sons, Rhys and Frey, who have each
gestated during the writing of two editions of this book. Finally, I would like to dedicate this edition to
the memory of my PhD mentor, Dr. Marietta Dunaway, a great inspiration who set my feet on
the exciting path of chromatin biology.
Jocelyn Krebs
To my family: my wife, Suzanne, whose patience, understanding, and confidence in me
are amazing; my children, Andy, Hyla, and Gary, who have taught me so much about
using the computer; and my grandchildren, Seth and Elena, whose smiles and giggles
inspire me. And to the memory of my mentor and dear friend, Lee A. Snyder, whose
professionalism, guidance, and insight demonstrated the skill.s necessary to be a scientist
and teacher. I have tried to live up to his expectations. This is for you, Doc.
Elliott Goldstein
To my wife, Lori, for our many years of love, support, and sometimes tolerance; to my
daughter, Jennifer, who will actually read this book; to my son, Andrew, who continually
renews my faith in humanity; and to my daughter, Sarah, who brings me joy daily.
Stephen Kilpatrick
6. Brief Table of Contents
Contents vii
Preface xix
About the Authors xxi
Part 1. GENES AND CHROMOSOMES 1
Chapter 1. Genes Are DNA 2
Chapter 2. Genes Encode RNAs and
Polypept1des 26
Chapter 3. Methods in Molecular B1ology and
Genetic Engineering 42
Chapter 4. The Interrupted Gene 81
Edited by Donald Forsdyke
Chapter 5. The Content of the Genome 100
Chapter 6. Genome Sequences and Gene
Numbers 120
Chapter 7. Clusters and Repeats 141
Chapter 8. Genome Evolut1on 161
Chapter 9. Chromosomes 192
Edited by Hank W. Bass
Chapter 10. Chromat1n 223
Part 2. DNA REPLICATION
AND RECOMBINATION 265
Chapter 11. Repl1cation Is Connected to
the Cell Cycle 266
Edited by Barbara Funnell
Chapter 12. The Replicon: In1t1at1on of
Repl1cat1on 286
Edited by Stephen D. Bell
Chapter 13. DNA Repl1cation 304
Edited by Peter Burgers
Chapter 14. Extrachromosomal Repl1cons 328
Edited by S�ren Johannes S�rensen and
Lars Hestbjerg Hansen
Chapter 15. Homologous and Site-Specific
Recomb1nat1on 354
Edited by Hannah L. Klein and Samantha Hoot
vi
Chapter 16. Repair Systems 395
Chapter 17. Transposable Elements and
Retrov1ruses 424
Edited by Damon Lisch
Chapter 18. Somatic Recomb1nat1on and
Hypermutat1on 1n the Immune System 459
Part 3. TRANSCRIPTION
AND POSTTRANSCRIPTIONAL
MECHANISMS 508
Chapter 19. Prokaryotic Transcr1pt1on 509
Edited by Richard Gourse
Chapter 20. Eukaryot1c Transcr1pt1on 549
Chapter 21. RNA Splicing and Processing 578
Chapter 22. mRNA Stab1l1ty and
Local1zat1on 622
Edited by Ellen Baker
Chapter 23. Catalyt1c RNA 647
Edited by Douglas J. Bn·ant
Chapter 24. Translat1on 671
Chapter 25. Us1ng the Genet1c Code 714
Edited by John Perona
Part 4. GENE REGULATION 744
Chapter 26. The Operon 745
Edited by Uskin Swint-Kruse
Chapter 27. Phage Strateg1es 777
Chapter 28. Eukaryot1c Transcr1pt1on
Regulat1on 804
Chapter 29. Ep1genet1c Effects Are
Inherited 838
Edited by Trygve Tollejsbol
Chapter 30. Regulatory RNA 872
Glossary 894
Index 920
7. Contents
Preface xix
About the Authors xxi
PART 1. GENES AND
CHROMOSOMES 1
Chapter 1. Genes Are DNA 2
111111 Introduction 3
liD DNA Is the Genetic Material of Bacteria
and Viruses 5
.a DNA Is the Genetic Material of Eukaryotic Cells 6
.a Polynucleotide Chains Have Nitrogenous Bases
Linked to a Sugar-Phosphate Backbone 7
.0 Supercoiling Affects the Structure of DNA 8
.m DNA Is a Double Helix 10
.U DNA Replication Is Semiconservative 12
.a Polymerases Act on Separated DNA Strands at
the Replication Fork 13
liB Genetic Information Can Be Provided by DNA
or RNA 14
Nucleic Acids Hybridize by Base Pairing 15
Mutations Change the Sequence of DNA 17
Mutations May Affect Single Base Pairs or
Longer Sequences 18
The Effects of Mutations Can Be Reversed 19
Mutations Are Concentrated at Hotspots 20
Many Hotspots Result from Modified Bases 21
Some Hereditary Agents Are Extremely Small 22
Summary 23
References 24
Chapter 2. Genes Encode RNAs
and Polypeptides 26
0::. Introduction 27
UJ1 Most Genes Encode Polypeptides 28
U. Mutations in the Same Gene Cannot
Complement 29
lfD Mutations May Cause Loss of Function or
Gain of Function 30
.0 A Locus May Have Many Different Mutant
Alleles 31
.a A Locus May Have More Than One Wild-Type
Allele 31
U. Recombination Occurs by Physical Exchange
of DNA 32
&a The Genetic Code Is Triplet 34
Every Coding Sequence Has Three Possible
Reading Frames 36
W:.St•l Bacterial Genes Are Colinear with Their
Products 37
W:.SII Several Processes Are Required to Express
the Product of a Gene 38
Proteins Are trans-Acting but Sites on
DNA Are ds-Acting 39
1':.'851 Summary 41
References 41
Chapter 3. Methods in Molecular Biology
and Genetic Engineering 42
0. Introduction 43
Ha Nucleases 44
UJI Cloning 46
..vn
8. liB Cloning Vectors Can Be Specialized for
Different Purposes 49
liD Nucleic Acid Detection 52
I!I'JI DNA Separation Techniques 54
DNA Sequencing 58
II!JI PCR and RT-PCR 59
liB Blotting Methods 65
Wlt•l DNA Microarrays 69
Will Chromatin lmmunoprecipitation 72
Wlfa Gene Knockouts and Transgenics 74
Will Summary 79
Chapter 4. The Interrupted Gene 81
Edited by Donald Forsdyke
Bll Introduction 82
UJI An Interrupted Gene Consists of Exons
and lntrons 83
&II Exon and lntron Base Compositions Differ 84
11!91 Organization of Interrupted Genes May Be
Conserved 84
Exon Sequences Under Negative Selection Are
Conserved but lntrons Vary 86
Exon Sequences Under Positive Selection Vary but
lntrons Are Conserved 87
Genes Show a Wide Distribution of Sizes
Due Primarily to lntron Size and Number
Variation 88
BJI Some DNA Sequences Encode More Than One
Polypeptide 90
&:. Some Exons Can Be Equated with Protein Functional
Domains 92
Hl•l Members of a Gene Family Have a Common
Organization 93
11111 There Are Many Forms of Information in DNA 95
Hfa Summary 97
References 97
viii Contents
Chapter 5. The Content of the Genome 100
&II Introduction 101
HJI Genomes Can Be Mapped at Several levels of
Resolution 102
.. Individual Genomes Show Extensive Variation 103
Hll RFLPs and SNPs Can Be Used for Genetic
Mapping 104
Bll Eukaryotic Genomes Contain Nonrepetitive and
Repetitive DNA Sequences 106
BiJI Eukaryotic Protein-Coding Genes Can Be Identified
by the Conservation of Exons 107
U. The Conservation of Genome Organization Helps to
Identify Genes 109
U. Some Organelles Have DNA 111
U. Organelle Genomes Are Circular DNAs That Encode
Organelle Proteins 113
10"11•1 The Chloroplast Genome Encodes Many Proteins
and RNAs 115
10"111 Mitochondria and Chloroplasts Evolved
by Endosymbiosis 116
IO"Ifa Summary 117
References 117
Chapter 6. Genome Sequences and
Gene Numbers 120
011 Introduction 121
tmfa Prokaryotic Gene Numbers Range Over
an Order of Magnitude 122
U. Total Gene Number Is Known for Several
Eukaryotes 123
lrD How Many Different Types of Genes Are There? 125
11'D The Human Genome Has Fewer Genes Than Originally
Expected 127
Ira How Are Genes and Other Sequences Distributed in
the Genome? 129
0. The Y Chromosome Has Several Male-Specific
Genes 131
0. How Many Genes Are Essential? 132
9. 0.. About 10,000 Genes Are Expressed at Widely
Differing Levels in a Eukaryotic Cell 135
Ut•l Expressed Gene Number Can Be Measured
En Masse 136
li!UI Summary 137
References 138
Chapter 7. Clusters and Repeats 141
16111 Introduction 142
liD Unequal Crossing Over Rearranges Gene
Clusters 144
U. Genes for rRNA Form Tandem Repeats Including an
Invariant Transcription Unit 147
Crossover Fixation Could Maintain Identical
Repeats 149
Satellite DNAs Often Lie in Heterochromatin
Arthropod Satellites Have Very Short Identical
Repeats 154
Mammalian Satellites Consist of Hierarchical
Repeats 154
Minisatellites Are Useful for Genetic Mapping
U. Summary 159
References 160
Chapter 8. Genome Evolution 161
Introduction 162
152
158
.0 DNA Sequences Evolve by Mutation and a Sorting
Mechanism 163
ED Selection Can Be Detected by Measuring Sequence
Variation 165
.0 A Constant Rate of Sequence Divergence Is a
Molecular Clock 169
ED The Rate of Neutral Substitution Can Be Measured
from Divergence of Repeated Sequences 173
liD How Did Interrupted Genes Evolve? 174
lila Why Are Some Genomes So Large? 177
�� Morphological Complexity Evolves by Adding New
Gene Functions 179
liB Gene Duplication Contributes to Genome
Evolution 180
Globin Clusters Arise by Duplication and
Divergence 181
Pseudogenes Are Nonfunctional Gene Copies 183
Genome Duplication Has Played a Role in Plant and
Vertebrate Evolution 185
What Is the Role of Transposable Elements in
Genome Evolution? 186
There May Be Biases in Mutation, Gene Conversion,
and Codon Usage 187
Summary 188
References 189
Chapter 9. Chromosomes 192
Edited by Hank W. Bass
U. Introduction 193
ua
a.
�
.a
�
Viral Genomes Are Packaged into Their Coats 194
The Bacterial Genome Is a Nucleoid 197
The Bacterial Genome Is Supercoiled 198
Eukaryotic DNA Has Loops and Domains Attached to
a Scaffold 200
Specific Sequences Attach DNA to an Interphase
Matrix 201
U. Chromatin Is Divided into Euchromatin and
Heterochromatin 202
0. Chromosomes Have Banding Patterns 204
� Lampbrush Chromosomes Are Extended 205
Polytene Chromosomes Form Bands 206
Polytene Chromosomes Expand at Sites of Gene
Expression 208
The Eukaryotic Chromosome Is a Segregation
Device 209
Regional Centromeres Contain a Centromeric Histone
H3 Variant and Repetitive DNA 210
Point Centromeres inS. cerevisiae Contain Short,
Essential DNA Sequences 212
The S. cerevisiae Centromere Binds a Protein
Complex 213
Telomeres Have Simple Repeating Sequences 213
Telomeres Seal the Chromosome Ends and Function
in Meiotic Chromosome Pairing 214
Contents ix
10. lill:l Telomeres Are Synthesized by a Ribonucleoprotein
Enzyme 216
li8Cil Telomeres Are Essential for Survival 218
0..1•1 Summary 219
References 220
Chapter 10. Chromatin 223
11•81 Introduction 225
DNA Is Organized in Arrays of Nucleosomes 225
11•11 The Nucleosome Is the Subunit of ALL
Chromatin 228
Nucleosomes Are Covalently Modified 231
II•JIO.. Histone Variants Produce Alternative
Nucleosomes 234
ll•!iW DNA Structure Varies on the Nucleosomal
Surface 237
The Path of Nucleosomes in the Chromatin
Fiber 240
11•!:1 Replication of Chromatin Requires Assembly of
Nucleosomes 242
II•Xil Do Nucleosomes Lie at Specific Positions? 245
11•81•1 Nucleosomes Are Displaced and
Reassembled During Transcription 249
lle811 DNase Sensitivity Detects Changes in
Chromatin Structure 252
ll•8fJ Insulators Define Transcriptionally Independent
Domains 254
11•80 An LCR May Control a Domain 258
11•8[1 Summary 260
References 261
PART 2. DNA REPLICATION
AND RECOMBINATION 265
Chapter 11. Replication Is Connected to
the Cell Cycle 266
Edited by Barbara Funnell
1181 Introduction 267
lllfM Bacterial Replication Is Connected to the Cell
Cycle 269
x Contents
1181 The Septum Divides a Bacterium into Progeny That
Each Contain a Chromosome 270
Mutations in Division or Segregation Affect Cell
Shape 271
11110.. FtsZ Is Necessary for Septum Formation 272
110 min and nocjslm Genes Regulate the Location of
the Septum 273
Chromosomal Segregation May Require Site-Specific
Recombination 274
111:1 Partition Involves Separation of the
Chromosomes 276
IIIII The Eukaryotic Growth Factor Signal Transduction
Pathway 277
Checkpoint Control for Entry Into S Phase: p53, a
Guardian of the Checkpoint 280
Checkpoint Control for Entry into S Phase: Rb, a
Guardian of the Checkpoint 281
Summary 282
References 283
Chapter 12. The Replicon: Initiation of
Replication 286
Edited by Stephen D. Bell
Introduction 287
An Origin Usually Initiates Bidirectional
Replication 288
lf&l The Bacterial Genome Is (Usually) a Single Circular
Replicon 289
Methylation of the Bacterial Origin Regulates
Initiation 290
If&.. Initiation: Creating the Replication Forks at the
Origin oriC 291
lfU Multiple Mechanisms Exist to Prevent Premature
Reinitiation of Replication 294
Archaeal Chromosomes Can Contain Multiple
Replicons 295
lfJ:I Each Eukaryotic Chromosome Contains Many
Replicons 295
lf<lfl Replication Origins Can Be Isolated in Yeast 297
Licensing Factor Controls Eukaryotic
Rereplication 298
11. lf-811 Licensing Factor Binds to ORC 299
lf.WfJ Summary 301
References 302
Chapter 13. DNA Replication 304
Edited by Peter Burgers
15!11 Introduction 305
15!'<1 DNA Polymerases Are the Enzymes That Make
DNA 306
15111 DNA Polymerases Have Various Nuclease
Activities 308
DNA Polymerases Control the Fidelity of
Replication 308
1&-1 DNA Polymerases Have a Common Structure 310
15!!1 The Two New DNA Strands Have Different Modes of
Synthesis 311
Replication Requires a Helicase and a Single-Strand
Binding Protein 312
15!:1 Priming Is Required to Start DNA Synthesis 313
15JCil Coordinating Synthesis of the Lagging and
Leading Strands 314
15!11•1 DNA Polymerase Holoenzyme Consists of
Subcomplexes 315
15!111 The Clamp Controls Association of Core Enzyme
with DNA 316
15Jifj Okazaki Fragments Are Linked by Ligase 319
15101 Separate Eukaryotic DNA Polymerases Undertake
Initiation and Elongation 321
I!Jit1 Lesion Bypass Requires Polymerase
Replacement 323
15!1101 Termination of Replication 325
15Jif!i Summary 325
References 326
Chapter 14. Extrachromosomal
Replicons 328
Edited by Seren Johannes Serensen and Lars Hestbjerg Hansen
lt.WI Introduction 329
lt*<l The Ends of Linear DNA Are a Problem for
Replication 330
1$1 Terminal Proteins Enable Initiation at the Ends of
Viral DNAs 331
Rolling Circles Produce Multimers of a
Replicon 332
lt$-1 Rolling Circles Are Used to Replicate Phage
Genomes 334
ltf!W The F Plasmid Is Transferred by Conjugation
Between Bacteria 335
Conjugation Transfers Single-Stranded DNA 336
lt.J:I Single-Copy Plasmids Have a Partitioning
System 338
lt11.!1 Plasmid Incompatibility Is Determined by the
Replicon 340
The ColE1 Compatibility System Is Controlled by
an RNA Regulator 341
How Do Mitochondria Replicate and
Segregate? 343
D Loops Maintain Mitochondrial Origins 344
The Bacterial Ti Plasmid Causes Crown Gall Disease
in Plants 346
T-DNA Carries Genes Required for Infection 347
Transfer of T-DNA Resembles Bacterial
Conjugation 349
Summary 351
References 352
Chapter 15. Homologous and Site-Specific
Recombination 354
Edited by Hannah L. Klein and Samantha Hoot
110"11 Introduction 356
110"!'<1 Homologous Recombination Occurs Between
Synapsed Chromosomes in Meiosis 358
110"11 Double-Strand Breaks Initiate Recombination 359
110"1'1 Gene Conversion Accounts for Interallelic
Recombination 361
110110-1 The Synthesis-Dependent Strand-Annealing
Model 363
110-!il The Single-Strand Annealing Mechanism Functions
at Some Double-Strand Breaks 364
Break-Induced Replication Can Repair Double-Strand
Breaks 364
Contents xi
12. 110-!;l Recombining Meiotic Chromosomes Are Connected
by the Synaptonemal Complex 365
11011il The Synaptonemal Complex Forms After
Double-Strand Breaks 367
11011(•) Pairing and Synaptonemal Complex Formation Are
Independent 369
1101111 The Bacterial RecBCD System Is Stimulated by
chiSequences 370
11011f.l Strand-Transfer Proteins Catalyze Single-Strand
Assimilation 371
1101UI Holliday Junctions Must Be Resolved 374
11011H Eukaryotic Genes Involved in Homologous
Recombination 376
11011101 Specialized Recombination Involves Specific
Sites 379
110110 Site-Specific Recombination Involves Breakage and
Reunion 380
110110 Site-Specific Recombination Resembles
Topoisomerase Activity 381
110111;1 Lambda Recombination Occurs in an Intasome 383
110110 Yeast Can Switch Silent and Active Loci for
Mating Type 384
1101fl•J Unidirectional Gene Conversion Is Initiated by
the Recipient MAT Locus 386
11011'.11 Antigenic Variation in Trypanosomes Uses
Homologous Recombination 387
11011''1'.1 Recombination Pathways Adapted for
Experimental Systems 388
11011'11 Summary 391
References 392
Chapter 16. Repair Systems 395
lti!il Introduction 396
lli!f.l Repair Systems Correct Damage to DNA 398
11$1 Excision Repair Systems in f. Coli 400
l(iJt'l Eukaryotic Nucleotide Excision Repair
Pathways 401
lti!0-1 Base Excision Repair Systems Require
Glycosylases 404
ltD Error-Prone Repair and Translesion Synthesis 406
xii Contents
Controlling the Direction of Mismatch Repair 407
l(i!;l Recombination-Repair Systems in f. coli 409
ltilil Recombination Is an Important Mechanism to
Recover from Replication Errors 411
lti!il•J Recombination-Repair of Double-Strand Breaks in
Eukaryotes 413
lti!Jill Nonhomologous End-Joining Also Repairs Double
Strand Breaks 414
lti!if.l DNA Repair in Eukaryotes Occurs in the Context of
Chromatin 415
lti!ill RecA Triggers the SOS System 418
lti!Ut Summary 420
References 420
Chapter 17. Transposable Elements
and Retroviruses 424
Edited by Damon Lisch
Ifill Introduction 426
lflf.l Insertion Sequences Are Simple Transposition
Modules 427
lf&l Transposition Occurs by Both Replicative and
Nonreplicative Mechanisms 429
Transposons Cause Rearrangement of DNA 430
lfJI0-1 Replicative Transposition Proceeds Through a
Cointegrate 431
lfJtiW Nonreplicative Transposition Proceeds by
Breakage and Reunion 433
Transposons Form Superfamilies and Families 434
IQ;I The Role of Transposable Elements in
Hybrid Dysgenesis 437
lfAil P Elements Are Activated in the Germline 438
Ifill•) The Retrovirus Lifecycle Involves Transposition-Like
Events 440
lfllll Retroviral Genes Code for Polyproteins 441
lfllf.l Viral DNA Is Generated by Reverse
Transcription 442
lfllll Viral DNA Integrates into the Chromosome 445
lfllh Retroviruses May Transduce Cellular Sequences 446
lflll01 Retroelements Fall into Three Classes 448
13. lfJi(!J Yeast Ty Elements Resemble Retroviruses 450
lfllifJ The Alu Family Has Many Widely Dispersed
Members 452
lfllt:l UNEs Use an Endonuclease to Generate a
Priming End 452
Summary 454
References 456
Chapter 18. Somatic Recombination and
Hypermutation in the Immune System 459
11:11 The Immune System: Innate and Adaptive
Immunity 462
lt:Jf<l The Innate Response Utilizes Conserved Recognition
Molecules and Signaling Pathways 463
lt:JJI Adaptive Immunity 466
Clonal Selection Amplifies Lymphocytes That
Respond to a Given Antigen 468
11:!0-1 lg Genes Are Assembled from Discrete DNA Segments
in B Lymphocytes 469
ll:!il L Chains Are Assembled by a Single Recombination
Event 471
H Chains Are Assembled by Two Sequential
Recombination Events 472
Recombination Generates Extensive Diversity 473
ll:fl V(D)J DNA Recombination Uses RSS and Occurs by
Deletion or Inversion 475
lt:ll•l Allelic Exclusion Is Triggered by Productive
Rearrangements 476
ft:lll RAG1/RAG2 Catalyze Breakage and Religation of
V(D)J Gene Segments 478
lt:lfJ B Cell Differentiation: Early lgH Chain Expression Is
Modulated by RNA Processing 480
lt:lll Class Switching Is Effected by DNA Recombination
(Class Switch DNA Recombination [CSR]) 481
lt:ltJj CSR Involves AID and Elements of the NHEJ
Pathway 483
11:1101 Somatic Hypermutation (SHM) Generates Additional
Diversity 484
lt:lt!J SHM Is Mediated by AID, Ung, Elements of the
Mismatch DNA Repair (MMR) Machinery, and
Translesion DNA Synthesis (TLS) Polymerases 486
Chromatin Modification in V(D)J Recombination,
CSR, and SHM 487
Expressed lgs in Avians Are Assembled from
Pseudogenes 488
B Cell Differentiation in the Bone Marrow and the
Periphery: Generation of Memory B Cells Enables a
Prompt and Strong Secondary Response 489
lt:Jfl•l The T Cell Receptor for Antigen (TCR) Is Related to
the BCR 492
lt:Jf.U The TCR Functions in Conjunction with the MHC 493
lt:JfJJ The Major Histocompatibility Complex (MHC) Locus
Comprises a Cohort of Genes Involved in Immune
Recognition 495
lt:Jf£1 Summary 498
References 499
PART 3. TRANSCRIPTION
AND POSTTRANSCRIPTIONAL
MECHANISMS 508
Chapter 19. Prokaryotic
Transcription 509
Edited by Richard Gourse
Introduction 511
Transcription Occurs by Base Pairing in a "Bubble"
of Unpaired DNA 512
lUI The Transcription Reaction Has Three Stages 513
Bacterial RNA Polymerase Consists of Multiple
Subunits 515
ICi)I0-1 RNA Polymerase Holoenzyme Consists of the Core
Enzyme and Sigma Factor 516
ICiD How Does RNA Polymerase Find Promoter
Sequences? 517
The Holoenzyme Goes Through Transitions in
the Process of Recognizing and Escaping from
Promoters 518
ICi!:l Sigma Factor Controls Binding to DNA by
Recognizing Specific Sequences in Promoters 519
llill!l Promoter Efficiencies Can Be Increased or Decreased
by Mutation 521
1Ci8l•l Multiple Regions in RNA Polymerase Directly Contact
Promoter DNA 523
Contents xiii
14. 1Ci811 RNA Polymerase-Promoter and DNA-Protein
Interactions Are the Same for Promoter Recognition
and DNA Melting 525
ICilfJ Interactions Between Sigma Factor and Core RNA
Polymerase Change During Promoter Escape 528
1Ci80 A Model for Enzyme Movement Is Suggested by the
Crystal Structure 529
ICi8tJ A Stalled RNA Polymerase Can Restart 531
1Ci810j Bacterial RNA Polymerase Terminates at Discrete
Sites 531
1Ci80 How Does Rho Factor Work? 533
ICilfJ Supercoiling Is an Important Feature of
Transcription 535
1Ci8t:l Phage T7 RNA Polymerase Is a Useful Model
System 536
ICi8CiJ Competition for Sigma Factors Can Regulate
Initiation 537
ICiJfl•J Sigma Factors May Be Organized into Cascades 539
ICiJfJI Sporulation Is Controlled by Sigma Factors 540
ICiJfiJ Antitermination Can Be a Regulatory Event 542
ICiJfD Summary 544
References 545
Chapter 20. Eukaryotic Transcription 549
Introduction 550
Eukaryotic RNA Polymerases Consist of Many
Subunits 552
RNA Polymerase I Has a Bipartite Promoter 554
RNA Polymerase III Uses Downstream and
Upstream Promoters 555
Wl•JIO"I The Start Point for RNA Polymerase II 557
Wl•!il TBP Is a Universal Factor 558
The Basal Apparatus Assembles at the
Promoter 561
Wl•!:l Initiation Is Followed by Promoter Clearance and
Elongation 564
Wl•Xil Enhancers Contain Bidirectional Elements That
Assist Initiation 567
fl•8l•J Enhancers Work by Increasing the Concentration of
Activators Near the Promoter 568
xiv Contents
fl•811 Gene Expression Is Associated with
Demethylation 569
fl•8fJ CpG Islands Are Regulatory Targets 571
fl•80 Summary
References 574
Chapter 21.
Processing
573
RNA Splicing and
578
Introduction 580
WJif. The 5' End of Eukaryotic mRNA Is Capped 581
WJ81 Nuclear Splice Sites Are Short Sequences 583
Splice Sites Are Read in Pairs 584
WJ&-1 Pre-mRNA Splicing Proceeds Through a Lariat 585
WJKil snRNAs Are Required for Splicing 586
Commitment of Pre-mRNA to the Splicing
Pathway 588
WJII:I The Spliceosome Assembly Pathway 591
WJIJ!I An Alternative Spliceosome Uses Different snRNPs
to Process the Minor Class of Introns 593
Pre-mRNA Splicing Likely Shares the Mechanism
with Group II Autocatalytic Introns 594
Splicing Is Temporally and Functionally Coupled
with Multiple Steps in Gene Expression 596
Alternative Splicing Is a Rule, Rather Than
an Exception, in Multicellular Eukaryotes 598
Splicing Can Be Regulated by Exonic and Intronic
Splicing Enhancers and Silencers 601
trans-Splicing Reactions Use Small RNAs 602
The 3' Ends of mRNAs Are Generated by Cleavage
and Polyadenylation 605
3' mRNA End Processing Is Critical for Termination
of Transcription 607
The 3' End Formation of Histone mRNA Requires
U7 snRNA 608
tRNA Splicing Involves Cutting and Rejoining in
Separate Reactions 609
The Unfolded Protein Response Is Related to tRNA
Splicing 612
15. f.llfl•J Production of rRNA Requires Cleavage Events and
Involves Small RNAs 613
f.llf.ll Summary 616
References 617
Chapter 22. mRNA Stability
and Localization 622
Edited by Ellen Baker
Introduction 623
Messenger RNAs Are Unstable Molecules 625
1'1&1 Eukaryotic mRNAs Exist in the Form of mRNPs from
Their Birth to Their Death 626
l'llf..W Prokaryotic mRNA Degradation Involves Multiple
Enzymes 627
1'14-1 Most Eukaryotic mRNA Is Degraded via Two
Deadenylation-Dependent Pathways 629
l'l.lil Other Degradation Pathways Target Specific
mRNAs 631
mRNA-Specific Half-Lives Are Controlled by
Sequences or Structures Within the mRNA 633
I'IJ:I Newly Synthesized RNAs Are Checked for Defects via
a Nuclear Surveillance System 635
1'1.&!1 Quality Control of mRNA Translation Is Performed by
Cytoplasmic Surveillance Systems 636
fi.Wl•J Translationally Silenced mRNAs Are Sequestered in a
Variety of RNA Granules 639
fi.WII Some Eukaryotic mRNAs Are Localized to Specific
Regians of a Cell 640
fi.WfJ Summary 643
References 644
Chapter 23. Catalytic RNA 647
Edited by Douglas J. Briant
I'JII Introduction 648
I'Jifa Group I lntrons Undertake Self-Splicing by
Transesterification 649
I'DI Group I lntrons Form a Characteristic Secondary
Structure 651
I'J!..W Ribozymes Have Various Catalytic Activities 653
I'JI0-1 Some Group I Introns Encode Endonucleases That
Sponsor Mobility 655
I'J!il Group II lntrons May Encode Multifunction
Proteins 657
Some Autosplicing Introns Require Maturases 658
The Catalytic Activity of RNase P Is Due to
RNA 659
I'JK!I Viroids Have Catalytic Activity 659
RNA Editing Occurs at Individual Bases 661
RNA Editing Can Be Directed by Guide RNAs 663
Protein Splicing Is Autocatalytic 665
Summary 667
References 667
Chapter 24. Translation 671
Introduction 673
I'.I.*a Translation Occurs by Initiation, Elongation, and
Termination 674
1'31 Special Mechanisms Control the Accuracy
of Translation 676
Initiation in Bacteria Needs 30S Subunits and
Accessory Factors 677
1'3-1 Initiation Involves Base Pairing Between mRNA and
rRNA 679
I'Ha A Special Initiator tRNA Starts the Polypeptide
Chain 680
Use of fMet-tRNA1 Is Controlled by IF-2 and the
Ribosome 681
l'.f.l!:l Small Subunits Scan for Initiation Sites on
Eukaryotic mRNA 683
l'.f.1Cil Eukaryotes Use a Complex of Many Initiation
Factors 684
Elongation Factor Tu Loads Aminoacyl-tRNA into
the A Site 687
The Polypeptide Chain Is Transferred to
Aminoacyl-tRNA 688
Translocation Moves the Ribosome 689
Elongation Factors Bind Alternately to
the Ribosome 690
Three Codons Terminate Translation 692
Termination Codons Are Recognized by Protein
Factors 693
Contents xv
16. f.t.'ltJ Ribosomal RNA Pervades Both Ribosomal
Subunits 694
f.t.'IU Ribosomes Have Several Active Centers 698
f.t.'lt:l 16S rRNA Plays an Active Role in Translation 700
23S rRNA Has Peptidyl Transferase Activity 702
Ribosomal Structures Change When the Subunits
Come Together 704
Translation Can Be Regulated 704
f.l.*..I'J The Cycle of Bacterial Messenger RNA 705
f.l.*£1 Summary 708
References 709
Chapter 25. Using the Genetic Code 714
Edited by John Perona
W-'10"11 Introduction 715
W-'10"1'.1 Related Codons Represent Chemically Similar
Amino Acids 716
W-'10111 Codon-Anticodon Recognition Involves
Wobbling 717
tRNAs Are Processed from Longer Precursors 719
W-'10"!0-1 tRNA Contains Modified Bases 720
W.<JOU Modified Bases Affect Anticodon-Codon Pairing 722
There Are Sporadic Alterations of the Universal
Code 723
Novel Amino Acids Can Be Inserted at Certain Stop
Codons 725
W-'10"111 tRNAs Are Charged with Amino Acids by Aminoacyl
tRNA Synthetases 726
f.<JO"Il•l Aminoacyl-tRNA Synthetases Fall into Two
Classes 728
f-'10"811 Synthetases Use Proofreading to Improve
Accuracy 730
f.<JO"IfJ Suppressor tRNAs Have Mutated Anticodons That
Read New Codons 732
fJ"IO There Are Nonsense Suppressors for Each
Termination Codon 734
f<101Ut Suppressors May Compete with Wild-Type Reading of
the Code 734
The Ribosome Influences the Accuracy of
Translation 736
xvi Contents
fJ"ItJ Frameshifting Occurs at Slippery Sequences 738
fJ"IU Other Recoding Events: Translational Bypassing and
the tmRNA Mechanism to Free Stalled
Ribosomes 740
fJ"It:l Summary 741
References 742
PART 4. GENE REGULATION 744
Chapter 26. The Operon 745
Edited by Liskin Swint-Kruse
W.tilil Introduction 747
WWM Structural Gene Clusters Are Coordinately
Controlled 750
W31 The lac Operon Is Negative Inducible 751
W.Mt.il lac Repressor Is Controlled by a Small-Molecule
Inducer 752
W.ti!0-1 as-Acting Constitutive Mutations Identify
the Operator 754
W.li!il trans-Acting Mutations Identify the Regulator
Gene 754
lac Repressor Is a Tetramer Made of Two Dimers 756
W.t!'!:l lac Repressor Binding to the Operator Is Regulated
by an Allosteric Change in Conformation 758
W.fiKil lac Repressor Binds to Three Operators and Interacts
with RNA Polymerase 759
f.tilil•l The Operator Competes with Low-Affinity Sites to
Bind Repressor 760
f.tilill The lac Operon Has a Second Layer of Control:
Catabolite Repression 762
f.tilifJ The trp Operon Is a Repressible Operon with Three
Transcription Units 764
f.(;i!UI The trp Operon Is Also Controlled by
Attenuation 766
f.(;ililiH Attenuation Can Be Controlled by Translation 767
f.tilil01 Stringent Control by Stable RNA Transcription 770
f.tilitJ r-Protein Synthesis Is Controlled by
Autoregulation 771
f.tiliU Summary 773
References 774
17. Chapter 27. Phage Strategies 777
WBII Introduction 779
Wi1t.J Lytic Development Is Divided into Two Periods 780
WIAI Lytic Development Is Controlled by a Cascade 781
Two Types of Regulatory Events Control the Lytic
Cascade 782
W&., The Phage T7 and T4 Genomes Show Functional
Clustering 783
WDfl Lambda Immediate Earty and Delayed Earty Genes Are
Needed for Both Lysogeny and the Lytic Cycle 784
The Lytic Cycle Depends on Antitermination by
pN 785
WU:I Lysogeny Is Maintained by the Lambda Repressor
Protein 787
WJWI The Lambda Repressor and Its Operators Define the
Immunity Region 788
fiJi(•) The DNA-Binding Form of the Lambda Repressor Is a
Dimer 788
fillII Lambda Repressor Uses a Helix-Turn-Helix Motif to
Bind DNA 790
fJJifJ Lambda Repressor Dimers Bind Cooperatively to the
Operator 791
Lambda Repressor Maintains an Autoregulatory
Circuit 792
fJIItJ Cooperative Interactions Increase the Sensitivity of
Regulation 793
fllll01 The ell and clll Genes Are Needed to Establish
Lysogeny 795
A Poor Promoter Requires ell Protein 796
fJJiQ Lysogeny Requires Several Events 796
fJJit:l The Cro Repressor Is Needed for Lytic Infection 798
fiJi@ What Determines the Balance Between Lysogeny
and the Lytic Cycle? 800
fi1tl•J Summary 801
References 802
Chapter 28. Eukaryotic Transcription
Regulation 804
W.<t:fll Introduction 805
W.<t:!'.J How Is a Gene Turned On? 807
W.<t:ll Mechanism of Action of Activators and
Repressors 808
Independent Domains Bind DNA and Activate
Transcription 811
W.<t:JIO., The Two-Hybrid Assay Detects Protein-Protein
Interactions 812
W.<t:U Activators Interact with the Basal Apparatus 813
There Are Many Types of DNA-Binding Domains 815
W.<t:!:l Chromatin Remodeling Is an Active Process 817
W.<t:Jt!l Nucleosome Organization or Content May Be
Changed at the Promoter 820
f.<t:fll•J Histone Acetylation Is Associated with Transcription
Activation 822
Methylation of Histones and DNA Is
Connected 825
Promoter Activation Involves Multiple Changes to
Chromatin 826
f.<t:flfil Histone Phosphorylation Affects Chromatin
Structure 827
Yeast GAL Genes: A Model for Activation and
Repression 829
Summary 831
References 833
Chapter 29. Epigenetic Effects Are
Inherited 838
Edited by Trygve To/lefsbol
Introduction 839
WDM Heterochromatin Propagates from a Nucleation
Event 841
W;Ji&l Heterochromatin Depends on Interactions with
Histones 842
Polycomb and Trithorax Are Antagonistic
Repressors and Activators 845
WU., X Chromosomes Undergo Global Changes 847
WUil Chromosome Condensation Is Caused by
Condensins 850
CpG Islands Are Subject to Methylation 853
WD:I DNA Methylation Is Responsible for
Imprinting 856
Contents xvii
18. W.JiXil Oppositely Imprinted Genes Can Be Controlled by a
Single Center 858
f.Jiii•J Epigenetic Effects Can Be Inherited 859
fUll Yeast Prions Show Unusual Inheritance 861
f.Ji)Jfj Prions Cause Diseases in Mammals 864
fUD summary 865
References 866
Chapter 30. Regulatory RNA 872
IJl•ll Introduction 873
IJl•!'a A Riboswitch Can Alter Its Structure According to
Its Environment 874
Wl•)l Noncoding RNAs Can Be Used to Regulate Gene
Expression 875
xviii Contents
Bacteria Contain Regulator RNAs 878
IJl•JI0-1 MicroRNAs Are Widespread Regulators in
Eukaryotes 881
Wl•!iW How Does RNA Interference Work? 884
Heterochromatin Formation Requires
MicroRNAs 887
IJl•!:l Summary 888
References 890
Glossary 894
Index 920
19. Preface
Of the diverse ways to study the living world, molecu
lar biology has been most remarkable in the speed and
breadth of its expansion. New data are acquired daily,
and new insights into well-studied processes come on a
scale measured in weeks or months rather than years.
It's difficult to believe that the first complete organis
mal genome sequence was obtained less than 20 years
ago. The structure and function of genes and genomes
and their associated cellular processes are sometimes
elegantly and deceptively simple but frequently amaz
ingly complex, and no single book can dojustice to the
realities and diversities of natural genetic systems.
This book is aimed at advanced students in molecu
lar genetics and molecular biology. In order to provide
the most current understanding of the rapidly chang
ing subjects in molecular biology, we have enlisted 21
scientists to provide revisions and content updates in
their individual fields of expertise. Their expert knowl
edge has been incorporated throughout the text. Much
of the revision and reorganization of this edition fol
lows that of the third edition of Lewin 's Essential GENES,
but there are many updates and features that are new
to this book. This edition follows a logical flow of top
ics; in particular, discussion of chromatin organization
and nucleosome structure precedes the discussion of
eukaryotic transcription, because chromosome organi
zation is critical to all DNA transactions in the cell, and
current research in the field of transcriptional regulation
is heavily biased toward the study of the role of chro
matin in this process. Many new figures are included
in this book, some reflecting new developments in the
field, particularly in the topics of chromatin structure
and function, epigenetics, and regulation by noncoding
and microRNAs in eukaryotes.
This book is organizedinto fourparts. Part 1 (Genes
and Chromosomes) comprises Chapters 1 through
10. Chapters 1 and 2 serve as an introduction to the
structure and function of DNA and contain basic cover
age of DNA replication and gene expression. Chapter 3
provides infotmation on molecular laboratory tech
niques. Chapter 4 introduces the interrupted structures
of eukaryotic genes, and Chapters 5 through 8 discuss
genome:: structure and evolution. Chapters 9 and 10 dis
cuss the structure of eukaryotic chromosomes.
Part2 (DNA Replication and Recombination)
comprises Chapters I I through 18. Chapters 1 1 to
14 provide detailed discussions of DNA replication
in plasmids, viruses, and prokaryotic and eukaryotic
cells. Chapters 15 through 1 8 cover recombination
and its roles in DNA repair and the human immune
system, with Chapter 16 discussing DNA repair path
ways in detail and Chapter 17 focusing on different
types of transposable elements.
Part 3 (Transcription and Posttranscriptional
Mechanisms) includes Chapters 19 through 25.
Chapters 19 and 20 provide more in-depth coverage
of bacterial and eukaryotic transcription. Chapters 21
through 23 are concerned with RNA, discussing mes
sengc::r RNA, RNA stability and localization, RNA pro
cessing, and the catalytic roles of RNA. Chapters 24 and
25 discuss translation and the genetic code.
Part 4 (Gene Regulation) comprises Chapters 26
through 30. In Chapter 26, the regulation of bacterial
gene:: expression via operons is discussed. Chapter 27
covc::rs the regulation of expression of genes during
phage:: development as they infect bacterial cells.
Chapters 28 and 29 cover eukaryotic gene regulation,
including epigenetic modifications. Finally, Chapter
30 covers RNA -based control of gene expression in
prokaryotes and eukaryotes.
For instructors who prefer to order topics with the
essc::ntials of DNA replication and gene expression fol
lowc::d by more advanced topics, the following chapter
sequc::nce is suggested:
Introduction: Chapters 1-2
Gc::ne and Genome Structure: Chapters 5-7
DNA Replication: Chapters 1 1-14
Transcription: Chapters 19-22
Translation: Chapters 24-25
Rc::gulation of Gene Expression: Chapters 9-10
and 26-30
Othc::r chapters can be covered at the instructor's
diseretion.
xix
20. Pedagogical Features
This edition contains several features to help students
learn as they read. Each chapter begins with a Chapter
Outline, and each section is summarized with a bul
leted list of Key Concepts. Key Terms are highlighted in
bold type in the text and compiled in the Glossary at
the end of the book. Finally, each chapter concludes
with an expanded and updated list of References,
which provides both primary literature and current
reviews to supplement and reinforce the chapter con
tent. Additional instructional tools are available on
line and on the Instructor's Media CD.
Ancillaries
Jones & Bartktt Learning offers an impressive array of
traditional and interactive multimedia supplements to
assist instructors and aid students in mastering molecu
lar biology. Additional information and review copies
of any of the following items are available through
your Jones & Bartlett sales representative or by visiting
http://www.jbleaming.com/science/molecular/.
For the Student
Interactive Student Study Guide
Jones & Bartlett Learning has developed an interac
tive, electronic study guide dedicated exclusively to
this title. Students will find a variety of study aids
and resources at http://biology.jbpub.com/lewin/
genesxiI, all designed to explore the concepts of
molecular biology in more depth and to help students
master the material in the book. A variety of activities
are available to help students review class material,
such as chapter summaries, Web-based learning exer
cises, study quizzes, a searchable glossary, and links to
animations, videos, and podcasts, all to help students
master important terms and concepts.
For Instructors
l11structor's Media CD
The Instructor's Media CD provides the instmctor with
the following resources:
• The PowerPoint• Image Bank provides all
of the illustrations, photographs, and tables
{to which Jones & Bartlett Learning holds the
copyright or has pennission to reprint digi-
xx Preface
tally) inserted into PowerPoint slides. With
the Microsoft• PowerPoint program, you can
quickly and easily copy individual image slides
into your existing lecture slides.
• The PowerPoint Lecture Outline presen
tation package developed by author Stephen
Kilpatrick, of the University of Pittsburgh at
Johnstown, provides outline summaries and
relevant images for each chapter of Lewin 's
GENES XI. A PowerPoint viewer is provided
on the CD, and instructors with the Microsoft
PowerPoint software can customize the out
lines, figures, and order of presentation.
Online l11structor Resources
The Test Bank, updated and expanded by author
Stephen Kilpatrick, is provided as a text file with 750
questions in a variety of formats. The Test Bank is easily
compatible with most course management software.
Acknowledgments
The authors would like to thank the following individ
uals for their assistance in the preparation of this book:
The editorial, production, marketing, and sales teams
at Jones & Bartlett have been exemplary in all aspects
of this proj ect. Cathy Sether, Erin O'Connor, Rachel
Isaacs, Lauren Miller, Leah Corrigan, and Wendy
Swanson deserve special mention. Cathy brought us
together on this proj ect and in doing so launched an
efficient and amiable partnership. She has provided
able leadership and has been an excellent resource
as we ventured into new territories. Rachel, Wendy,
and Leah have handled the daily responsibilities of the
writing and production phases with friendly profes
sionalism and helpful guidance. Lauren has made the
process of choosing and revising figures very smooth.
We thank the editors of individual chapters,
whose expertise, enthusiasm, and careful judgment
brought the manuscript up to date in many critical
areas.
Jocelyn E. Krebs
Elliott S. Goldstein
Stephen T. Kilpatrick
21. About the Authors
Benjamin Lewin founded the journal Cell in 1974
and was Editor until 1999. He founded the Cell Press
journals Neuron, Immunity, and Molecular Cell. In 2000,
he founded Virtual Text, which was acquired by Jones
and Bartlett Publishers in 2005. He is also the author
of Essmtial GENES and CELLS.
Jocelyn E. Krebs received a
B.A. in Biology from Bard Col
lege, Annandale-on-Hudson,
NY, and a Ph.D. in Molecular
and Cell Biology from the Uni
versity of California, Berkeley.
For her Ph.D. thesis, she stud
ied the roles of DNA topol
ogy and insulator elements in
transcriptional regulation.
She performed her post
doctoral training as an Ametican Cancer Society
Fellow at the University of Massachusetts Medical
School in the laboratory of Dr. Craig Peterson, where
she focused on the roles of histone acetylation and
chromatin remodeling in transniption. In 2000, Dr.
Krebs joined the faculty in the Department of Bio
logical Science� at the University of Alaska, Anchor
age, whc:re she is now an Associate Professor. She
directs a research group studying chromatin structure
and function in transcription and DNA repair in the
yeast Saccharomyces cerevisiae and the role of chroma
tin remodeling in embryonic development in the frog
Xenopus. She teaches courses in molecular biology
for undngraduates, graduate students, and first-year
medical students. She also teaches a Molecular Biol
ogy of Cancer course and has taught Genetics and
Introductory Biology. She lives in Eagle River, AK,
with her partner and son, and a house full of dogs and
cats. Her non-work passions include hiking, camping,
and snowshoeing.
EJijott S. Goldstein earned
his B.S. in Biology from the
University of Hartford (Con
necticut) and his Ph.D. in
Genetics from the University
of Minnesota, Department
of Genetics and Cell Biol
ogy. Following this, he was
awarded an N.I.H. Postdoc
toral Fellowship to work with
Dr. Sheldon Penman at the
Massachusetts Institute of Technology. After leaving
Boston, he joined the faculty at Arizona State Univer
sity in Tempe, AZ, where he is an Associate Professor
in the Cellular, Molecular, and Biosciences program
in the School of Life Sciences and in the Honors Dis
ciplinary Program. His research interests are in the
area of molecular and developmental genetics of early
embryogenesis in Drosophila melanogaster. In recent
years, he has focused on the Drosophila cow1terparts
of the human proto-oncogenes jun and fos. His pri
mary teaching responsibilities are in the undergradu
ate General Genetics course as well as the graduate
level Molecular Genetics course. Dr. Goldstein lives
in Tempe with his wife, his high school sweetheart.
They have three children and two grandchildren. He
is a bookwom1 who loves reading as well as underwa
ter photography. His pictures can be fmmd at http://
www.public.asu.edu/-elliotg/.
Stephen T. Kilpatrick
received a B.S. in Biology from
Eastern College (now Eastem
University) in St. Davids, PA,
and a Ph.D. from the Program in
Ecology and Evolutionary Biol
ogy at Brown University. His
thesis research was an investiga
tion of the population genetics
xxi
22. of interactions between the mitochondrial and nuclear
genomes of Drosophila melanogaster. Since 1995, Dr. Kilpat
rick has taught at the University of Pinsburgh at Johnstown
in Johnstown, PA. His regular teaching duties include
undergraduate courses in nonmajors biology, introduc
tory majors biology, and advanced undergraduate courses
in genetics, evolution, molecular genetics, and biostatis
tics. He has also supervised a number of undergraduate
research projects in evolutionary genetics. Dr. Kilpatrick's
major professional focus has been in biology education.
xxii About the Authors
He has participated in the development and authoring of
ancillary materials for several introductory biology, genet
ics, and molecular genetics texts as well as writing articles
for educational reference publications. For his classes at
Pitt-Johnstown, Dr. Kilpatrick has developed many active
learning exercises in introductory biology, genetics, and
evolution. Dr. Kilpal1ick resides in Johnstown, PA, with
his wife and three children. Outside of scientific interests,
he enjoys music, literature, and theater and occasionally
perfonns in local community theater groups.
23. Chapter Editors
Esther Siegfried completed her work on this book
while teaching at the University of Pittsburgh at John
stown. She is now an Assistant Professor of Biology at
Pennsylvania State University, Altoona. Her research
interests include signal transduction pathways in
Drosophila development.
John Brunstein is a Clinical Assistant Professor
with the Department of Pathology and Laboratory
Medicine at the University of British Columbia. His
research interests focus on the development of, vali
dation strategies for, and implementation of novel
molecular diagnostic technologies.
Donald Forsdyke, emeritus professor of bio
chemistry at Queen's University in Canada, studied
lymphocyte activation/inactivation and the associated
genes. In the 1990s he obtained evidence support
ing his 1981 hypothesis on the origin of introns, and
immunologists in Australia shared a Nobel Prize for
work that supported his 1975 hypothesis on the posi
tive selection of the lymphocyte repertoire. His books
include The Origin ofSpecies, Revisited (200 1 ), Evolution
ary Bioinformatics (2006 ), and "Treasure YourExceptions":
The Science and Lzfe of William Bateson (2008).
Hank W. Bass is an Associate Professor of Bio
logical Science at Florida State University. His labora
tory works on the stn1cture and function of meiotic
chromosomes and telomeres in maize using molecu
lar cytology and genetics.
Stephen D. Bell is the Professor of Microbiology
in the Sir William Dunn School of Pathology, Oxford
University. His research group is studying gene tran
scription, DNA replication, and cell division in the
Archaeal domain of life.
Seren Johannes Sarensen is a Professor in the
Department of Biology and Head of the Section of
Microbiology at the University of Copenhagen. The
main objective of his studies is to evaluate the extent
of genetic flow within natural communities and the
responses to environmental perturbations. Molecu
lar techniques such as DGGE and high-throughput
sequencing are used to investigate resilience and
resistance of microbial community structure. Dr
S0rensen has more than twenty years' experience in
teaching molecular microbiology at both the bachelor
and graduate levels.
Lars Hestbjerg Hansen is an Associate Professor
in the Section of Microbiology, Department of Biol
ogy, at the University of Copenhagen. His research
interests include the bacterial maintenance and
interchange of plasmid DNA, especially focused on
plasmidbome mechanisms of bacterial resistance to
antibiotics. Dr. Hansen's laboratory has developed and
is currently working with new flow-cytometric meth
ods for estimating plasmid transfer and stability. Dr.
Hansen is the Science Director of Prokaryotic Genom
ics at Copenhagen High-Throughput Sequencing
Facility, focusing on using high-throughput sequenc
ing to describe bacterial and plasmid diversity in natu
ral environments.
Barbara Funnell is a Professor of Molecular
Genetics at the University of Toronto. Her laboratory
studies chromosome dynamics in bacterial cells and,
in particular, the mechanisms of action of proteins
involved in plasmid and chromosome segregation.
Peter Burgers is Professor of Biochemistry and
Molecular Biophysics at Washington University
School of Medicine. His laboratory has a long-stand
ing interest in the biochemistry and genetics of DNA
replication in eukaryotic cells, and in the study of
responses to DNA damage and replication stress that
result in mutagenesis and in cell cycle checkpoints.
Hannah L. Klein is a Professor of Biochemistry,
Medicine, and Pathology at New York University Lan
gone Medical Center. She studies pathways of DNA
damage repair and recombination and genome stability.
Samantha Hoot is a postdoctoral researcher
in the laboratory of Dr. Hannah Klein at New York
University Langone Medical Center. She received her
Ph.D. from the University of Washington. Her interests
include the role of recombination in genome stability
in yeast and the molecular mechanisms of drug resis
tance in pathogenic fungi.
xxiii
24. Damon Lisch is an Associate Research Profes
sional at the University of California at Berkeley. He is
interested in the regulation of transposable elements
in plants and the ways in which tramposon activity
has shaped plant genome evolution. His laboratory
investigates the complex behavior and epigenetic reg
ulation of the Mutator system of transposons in maize
and related species.
Paolo casali, M.D., is the Donald L. Bren Professor
of Medicine, Molecular Biology & Biochemistry, and
Director of the Institute for Immunology at the Univer
sity of California, Irvine. He works on B lymphocyte
differentiation and regulation of antibody gene expres
sion as well as molecular mechanisms of generation of
autoantibodies. He has been Editor-in-Chief of Autoim
munity since 2002. He is a member of the American
Association of Immunologists, an elected "Young Turk"
of the American Society for Clinica1 Investigation, and
an elected Fellow of the American Association for
Advancement of Science. He has served on several NIH
immunology study sections and scientific panels.
Richard Gourse is a Professor in the Department
of Bacteriology at the University of Wisconsin, Madi
son, and an Editor of the Journal of Bacteriology. His
primary interests lie in transcription initiation and the
regulation of gene expression in bacteria. His labo
ratory has long focused on rRNA promoters and the
control of ribosome synthesis as a means of uncover
ing fundamental mechanisms responsible for regula
tion of transcription and translation.
Xiang-Dong Fu is a Professor of Cellular and
Molecular Medicine at the University of California,
San Diego. His laboratory studies mechanisms under
lying constitutive and regulated pre-mRNA splicing in
mammalian cells, coupling between transcription and
RNA processing, RNA genomics, and roles of RNA
processing in development and diseases.
Ellen Baker is an Associate Professor of Biol
ogy at the University of Nevada, Reno. Her research
xxiv Chapter Editors
interests have focused on the role of polyadenylation
in mRNA stability and translation.
Douglas J. Briant teaches at the University of
Victoria in British Columbia. His research has investi
gated bacterial RNA processing and the role of ubiqui
tin in cell signalling pathways.
Cheryl Keller Capone is an Instructor in the
Department of Biology at The Pennsylvania State
University and teaches cell and molecular biology. Her
research interests include embryonic muscle devel
opment in Drosophila melanogaster and the molecular
mechanisms involved in the clustering and postsyn
aptic targeting of GABAA receptors.
John Perona is a Professor of Biochemistry
in the Department of Chemistry and Biochemis
try, and the Interdepartmental Program in Biomo
lecular Science and Engineering, at the University
of California, Santa Barbara. His laboratory stud
ies stn1cture-function relationships and catalytic
mechanisms in aminoacyl-tRNA synthetases, tRNA
dependent amino acid modification enzymes, and
tRNA-modifying enzymes.
Liskin Swint-Kruse is an Assistant Professor in
Biochemistry and Molecular Biology at the University
of Kamas School of Medicine. Her research utilizes
bacterial transcription regulators in studies that bridge
biophysics of DNA-binding and bioinformatics analy
ses of transcription repressor families to advance the
principles of protein engineering.
Trygve Tollefsbol is a Professor of Biology at
the University of Alabama at Birmingham and a
Senior Scientist of the Center for Aging, Comprehen
sive Cancer Center, and Clinical Nutrition Research
Center. He has long been involved with elucidating
epigenetic mechanisms, especially as they pertain to
cancer, aging, and differentiation. He has been the
editor and primary contributor of numerous books
including Epigenetic Protocols, Cancer Epigenetics, and
Epigenetics ofAging.
29. Genes and Chromosomes
CHAPTER 1 Genes Are DNA
CHAPTER 2 Genes Encode RNAs and Polypeptides
CHAPTER3 Methods in Molecular Biology and Genetic
Engineering
CHAPTER 4 The Interrupted Gene
CHAPTER 5 The Content of the Genome
CHAPTER 6 Genome Sequences and Gene Numbers
CHAPTER 7 Clusters and Repeats
CHAPTER 8 Genome Evolution
CHAPTER 9 Chromosomes
CHAPTER 1 0 Chromatin
1
30. Genes Are DNA
CHAPTER OUTLINEJ
••• Introduction
••• DNA Is the Genetic Material of Bacteria and Viruses
• Bacterial transformation provided the first support
that DNA is the genetic material of bacteria. Genetic
properties can be transferred from one bacterial strain
to another by extracting DNA from the first strain and
adding it to the second strain.
• Phage infection showed that DNA is the genetic mate
rial of viruses. When the DNA and protein components
of bacteriophages are labeled with different radioac
tive isotopes, only the DNA is transmitted to the prog
eny phages produced by infecting bacteria.
••• DNA Is the Genetic Material of EukaJYotic Cells
2
• DNA can be used to introduce new genetic traits into
animalcells or whole animals.
• In some viruses, the genetic material is RNA.
Polynucleotide Chains Have Nitrogenous Bases
Linked to a Sugar-Phosphate Backbone
• A nucleoside consists of a purine or pyrimidine base
linked to the 1' carbon of a pentose sugar.
• The difference between DNA and RNA is in the
group at the 2' position ofthe sugar. DNA has a
deoxyribose sugar (2'-H); RNA has a ribose sugar
(2'-0H).
• A nucleotide consists of a nucleoside linked to a
phosphate group on either the 5 ' or 3 ' carbon ofthe
(deoxy)ribose.
• Successive (deoxy)ribose residues of a polynucleotide
chain are joined by a phosphate group between the
3' carbon of one sugar and the 5 ' carbon of the next
sugar.
• One end ofthe chain (conventionally written on the
left) has a free 5' end and the other end of the chain
has a free 3 ' end.
• DNA contains the four bases adenine, guanine, cyto
sine, and thymine; RNA has uracil instead ofthymine.
Supercoiling Affects the Structure of DNA
• Supercoiling occurs only in "closed" DNA with no free
ends.
• Closed DNA is either circular DNA or linear DNA in
which the ends are anchored so that they are not free
to rotate.
• A closed DNA molecule has a linking number (L), which
is the sum of twist (T) and writhe (W).
• The linking number can be changed only by breaking
and reforming bonds in the DNA backbone.
•n DNA Is a Double Helix
• The B-form of DNA is a double helix consisting of two
polynucleotide chains that run antiparallel.
31. CHAPTER OUTLINE, CONTINUEDJ
• The nitrogenous bases of each chain are flat purine or
pyrimidine rings that face inward and pair with one
another by hydrogen bonding to form only A-T or G-C
pa1rs.
• The diameter of the double helix is 20 A, and there is•
a complete turn every 34 A, with 10 base pairs per turn
(-10.4 base pairs per turn in solution).
• The double helix has a major (wide) groove and a
minor (narrow) groove.
DNA Replication Is Semiconservative
• The Meselson-Stahl experiment used "heayy" isotope
labeling to show that the single polynucleotide strand
is the unit of DNA that is conserved during replication.
• Each strand of a DNA duplex acts as a template for
synthesis of a daughter strand.
• The sequences ofthe daughter strands are determined
by complementary base pairing with the separated
parental strands.
.0 Polymerases Act on Separated DNA Strands at the
Replication Fork
• Replication of DNA is undertaken by a complex of
enzymes that separate the parental strands and
synthesize the daughter strands.
• The replication fork is the point at which the parental
strands are separated.
• The enzymes that synthesize DNA are called DNA
polymerases.
• Nucleases are enzymes that degrade nucleic acids; they
include DNases and RNases and can be categorized as
endonucleases or exonucleases.
Genetic Information Can Be Provided by DNA or RNA
• Cellular genes are DNA, but viruses may have genomes
of RNA.
• DNA is converted into RNA by transcription, and RNA
may be converted into DNA by reverse transcription.
• The translation of RNA into protein is unidirectional.
111•1 Nucleic Acids Hybridize by Base Pairing
• Heating causes the two strands of a DNA duplex to
separate.
• The Tm is the midpoint of the temperature range for
denaturation.
• Complementary single strands can renature when the
temperature is reduced.
• Denaturation and renaturation/hybridization can occur
with DNA-DNA, DNA-RNA, or RNA-RNA combinations
and can be intermolecular or intramolecular.
Bl Introduction
• The ability oftwo single-stranded nucleic acids to
hybridize is a measure oftheir complementarity.
1111 Mutations Change the Sequence of DNA
• All mutations are changes in the sequence of DNA.
• Mutations may occur spontaneously or may be induced
by mutagens.
llr.l Mutations May Affect Single Base Pairs or
Longer Sequences
• A point mutation changes a single base pair.
• Point mutations can be caused by the chemical
conversion of one base into another or by errors that
occur during replication.
• A transition replaces a G-C base pair with an A-T base
. .
pa1r orv1ce versa.
• A transversion replaces a purine with a pyrimidine,
such as changing A-T toT-A.
• Insertions andjor deletions can result from the
movement of transposable elements.
lid The Effects of Mutations Can Be Reversed
• Forward mutations alter the function of a gene, and
back mutations (or revertants) reverse their effects.
• Insertions can revert by deletion of the inserted
material, but deletions cannot revert.
• Suppression occurs when a mutation in a second gene
bypasses the effect of mutation in the first gene.
11$ Mutations Are Concentrated at Hotspots
• The frequency of mutation at any particular base pair
is statistically equivalent, except for hotspots, where
the frequency is increased by at least an order of
magnitude.
1110'1 Many Hotspots Result from Modified Bases
• A common cause of hotspots is the modified base
5-methylcytosine, which is spontaneously deaminated
to thymine.
• A hotspot can result from the high frequency of
change in copy number of a short, tandemly repeated
sequence.
IIMI Some Hereditary Agents Are Extremely Small
• Some very small hereditary agents do not encode
polypeptide, but consist of RNA or protein with
heritable properties.
lltl Summary
The hereditary basis of every living organism
is its genome, a long sequence of deoxyri
bonucleic acid (DNA) that provides the com·
plete set of hereditary information carried by
the organism as well as its individual cells.
The genome includes chromosomal DNA as
well as DNA in plasmids and (in eukaryotes)
organellar DNA, as found in mitochondria
and chloroplasts. We use the term informa·
tion because the genome does not itself per·
form an active role in the development of
1.1 Introduction 3
32. the organism. The products of expression
of nucleotide sequences within the genome
determine development. By a complex series
of interactions, the DNA sequence produces
all of the proteins of the organism at the
appropriate time and within the appropriate
cells. Proteins serve a diverse series of roles
in the development and functioning of an
organism; they can form part of the struc
ture of the organism, have the capacity to
build the structures, perform the metabolic
reactions necessary for life, and participate in
regulation as transcription factors, receptors,
key players in signal transduction pathways,
and other molecules.
Physically, the genome may be divided
into a number of different DNA molecules,
or chromosomes. The ultimate definition
of a genome is the sequence of the DNA of
each chromosome. Functionally, the genome
is divided into genes. Each gene is a sequence
ofDNA that encodes a single type of RNA and
in many cases, ultimately a polypeptide. Each
of the discrete chromosomes comprising the
genome may contain a large number of genes.
Genomes for living organisms may contain
as few as -500 genes (for a mycoplasma, a
type of bacterium), -20,000 to 25,000 for
a human being, or as many as -50,000 to
60,000 for rice.
In this chapter, we explore thegene in terms
of its basic molecular construction. FIGURE 1.1
1850
1900
1950
2000
1865 Genes are particulate factors
1871 Discovery of nucleic acids
1903 Chromosomes are hereditary units
1910 Genes lie on chromosomes
1913 Chromosomes are linear arrays of genes
1927 Mutations are physical changes in genes
1931 Recombination occurs by crossing over
1944 DNA is the genetic material
1945A gene codes for protein
1951 First protein sequence
........_1953 DNAis a double helix
1958 DNA replicates semiconservatively
1961 Genetic code is triplet
t--1977 Eukaryotic genes are interrupted
1977 DNA can be sequenced
1995 Bacterial genomes sequenced
2001 Human genome sequenced
FIGURE 1.1 A brief history of genetics.
4 CHAPTER 1 Genes Are DNA
Gene Chemical nature
DNA Sequence of nucleotides
RNA Sequence of nucleotides
Polypeptide � Sequence of amino acids
FIGURE 1.2 A gene encodes an RNA, which may encode
a polypeptide_
summarizes the stages in the transition from
the historical concept ofthegeneto the modern
definition of the genome.
The first definition of the gene as a func
tional unit followed from the discovery that
individual genes are responsible for the pro
duction of specificproteins. Later, the chemical
differences between the DNA of the gene and
its protein product led to the suggestion that a
gene encodes a protein. This in turn led to the
discovery of the complex apparatus by which
the DNA sequence of a gene determines the
amino acid sequence of a polypeptide.
Understanding the process by which a
gene is expressed allows us to make a more
rigorous definition of its nature. FIGURE 1.2
shows the basic theme of this book. A gene
is a sequence of DNA that directly produces
a single strand of another nucleic acid, RNA,
with a sequence that is identical to one of
the two polynucleotide strands of DNA. In
many cases, the RNA is in turn used to direct
production of a polypeptide. In other cases,
such as rRNA and tRNA genes, the RNA tran
scribed from the gene is the functional end
product. Thus, a gene is a sequence of DNA
that encodes an RNA, and in protein-coding,
or structural, genes, the RNA in turn encodes
a polypeptide.
From the demonstration that a gene con
sists of DNA, and that a chromosome consists
of a long stretch of DNA representing many
genes, we will move to the overall organiza
tion of the genome. In the chapter titled The
Interrupted Ge11e, we take up in more detail
the organization of the gene and its repre
sentation in proteins. In the chapter titled The
Content ofthe Genome, we consider the total
number of genes, and in the chapter titled
Clusters a11d Repeats, we discuss other compo
nents of the genome and the maintenance of
its organization.
33. Ill DNA Is the Genetic
Material of Bacteria
and Viruses
Key concepts
• Bacterial transformation provided the first support
that DNA is the genetic material of bacteria.
Genetic properties can be transferred from one
bacterial strain to another by extracting DNA from
the first strain and adding it to the second strain.
• Phage infection showed that DNA is the genetic
material of viruses. When the DNA and protein
components of bacteriophages are labeled with
different radioactive isotopes, only the DNA is
transmitted to the progeny phages produced by
infecting bacteria.
The idea that the genetic material of organ
isms is DNA has its roots in the discovery of
transformation by Frederick Griffith in 1928.
The bacterium Streptococcus (formerly PHeu
mococcus) pHeumoniae kills mice by causing
pneumonia. The virulence of the bacterium
is determined by its capsular polysaccharide,
which allows the bacterium to escape destruc
ti on by its host. Several types of S. p11eumo11iae
have different capsular polysaccharides, but
they all have a smooth (S) appearance. Each
of the S types can give rise to variants that fail
to produce the capsular polysaccharide and
therefore have a rough (R) surface (consisting
of the material that was beneath the capsu
lar polysaccharide). The R types are avirulent
and do not kill the mice because the absence of
the polysaccharide capsule allows the animal's
immune system to destroy the bacteria.
When S bacteria are killed by heat treat
ment, they can no longer harm the animal.
FIGURE 1.3, however, shows that when heat
killed S bacteria and avirulent R bacteria are
jointly injected into a mouse, it dies as the result
Pneumococcus types Injection of cells
Capsule Living S
smooth (S)
appearance
'""'-
Heat-killed S
Living R
No ca'l� Heat-killed S
rough (R) � Living R
appearance
Result
Die�
Live�
Live�
Die�
FIGURE 1.3 Neither heat-killed S-type nor live R-type
bacteria can kill mice, but simultaneous injection ofboth
can kill mice just as effectively as the live S type.
Mouse injected
with heat-killed S
and living R bacteria
. t;::�>
i Living S bacteria
recovered from
'--" dead mouse
Extract
DNA
��R b t . Transform
acena S bacteria
FIGURE 1.4 The DNA of S-type bacteria can transform
R-type bacteria into the same S type.
of a pneumonia infection. Virulent S bacteria
can be recovered from the mouse's blood.
In this experiment, the dead S bacteria
were of type III. The live R bacteria had been
derived from type IT. The virulent bacteria
recovered from the mixed infection had the
smooth coat of type III. So, some property of
the dead IllS bacteria can transform the live IIR
bacteria so that they make the capsularpolysac
charide and become virulent. FIGURE 1.4 shows
the identification of the component of the dead
bacteria responsible for transformation. This
was called the transformingprinciple. It was
purified in a cell-free system in which extracts
from the dead III$ bacteria were added to the
live IIR bacteria before beingplated on agarand
assayed for transformation (FIGURE 1.5). Puri
fication of the transforming principle in 1944
by Avery, MacLeod, and McCarty showed that
it is DNA.
FIGURE 1.5 Rough (left) and smooth (right) colonies of S. pneumoniae. �P Avery,
et al., 1944. Originally published in TheJournal ofExperimentalMedicine, 79: 137-158.
Used with permission ofThe Rockefeller University Press.
1.2 DNA Is the Genetic Material of Bacteria and Viruses 5
34. Infect bacteria with labeled
phages •
32
P in DNA
I •
35
8 in protein
Separate phage coats
and infected bacteria
1ItA:a:=/'--./=�
t (_ � � 'v--
Infected
bacteria
contain
70%of32
P label
Phage coats
contain 80% of I3s
s label Isolate progeny
phage particles
Progeny
phages
have 30%of
32
P label
and <1% of
35
S label
FIGURE 1.6 The genetic material of phage T2 is DNA.
Having shown that DNA is the genetic
material of bacteria, the next step was to
demonstrate that DNA is the genetic mate
rial in a quite different system. Phage T2 is a
virus that infects the bacterium Escherichia coli.
When phage particles are added to bacteria,
they attach to the outside surface, some mate
rial enters the cell, and then -20 minutes later
each cellbursts open, or lyses, to release a large
number of progeny phage.
FIGURE 1.6 illustrates the results ofan experi
ment conducted in 1952 by Alfred Hershey and
MarthaChaseinwhichbacteriawereinfectedwith
T2phagesthathadbeenradioactivelylabeledeither
in their DNA component (with phosphorus-32
[32P]) orin theirprotein component (with sulfur-
35 [35S]). The infected bacteria were agitated in
a blender, and two fractions were separated by
centrifugation. Onefraction containedthe empty
phage "ghosts" that were released from the sur
face ofthebacteria and the other consisted ofthe
infected bacteria themselves. Previously, it had
been shown that phage replication occurs intra
cellularly, sothatthegeneticmaterialofthephage
would have to enter the cell duringinfection.
Most ofthe 32P label waspresentin the frac
tion containing infected bacteria. The progeny
phage particles produced by the infection con
tained -30% of the original 32P label. The
progeny received less than 1% of the protein
contained in the originalphage population. The
phage ghosts consisted ofprotein and therefore
carried the 35S radioactive label. This experiment
6 CHAPTER 1 Genes Are DNA
directly showed that only the DNA ofthe parent
phages enters the bacteria and becomes part
of the progeny phages, which is exactly the
expected behavior of genetic material.
A phage reproducesby commandeeringthe
replication machinery ofan infected host cell to
manufacture more copies of itself. The phage
possesses geneticmaterial with properties anal
ogous to those ofcellulargenomes: Its traits are
faithfully expressed and are subjectto the same
rules that govern inheritance of cellular traits.
The case ofT2 reinforcesthe general conclusion
that DNA is the geneticmaterial of the genome
of a cell or a virus.
ID DNA Is the Genetic
Material of Eukaryotic
Cells
Key concepts
• DNA can be used to introduce new genetic traits
into animal cells or whole animals.
• In some viruses, the genetic material is RNA.
When DNA is added to eukaryotic cells grow
ing in culture, it can enter the cells, and in
some of them this results in the production of
new proteins. When an isolated gene is used,
its incorporation leads to the production of a
particular protein, as depicted in FIGURE 1.7.
Cells that lack TK gene cannot produce
thymidine kinase and die in absence of thymidine
• •
• •
•
• • •
Colony of ---=-.r-,::::-
TK+ cells
8
Dead cells
8
Live cells
Some cells take up TK gene; descendants of
transfected cell pile up into a colony
FIGURE 1.7 Eukaryoticcells can acquire a new phenotype
as the result oftransfection by added DNA.
35. Although for historical reasons these experi
ments are described as transfection when per
formed with animal cells, they are analogous to
bacterial transformation. The DNA thatis intro
duced into the recipient cell becomes part ofits
genome and isinherited with it, and expression
of the new DNA results in a new trait upon the
cells (synthesisof thymidine kinase in the exam
ple of Figure 1.7). At first, these experiments
were successful only wi th individual cellsgrow
ingin culture, but in laterexperimentsDNAwas
introduced into mouse eggs by microinjection
and became a stable part of the genome of the
mouse. Such experiments show directly that
DNA is the genetic material in eukaryotes and
that it can be transferred between different spe
cies and remain functional.
The genetic material of all known organ
isms and many viruses is DNA. Some viruses,
though, use RNA as the genetic material. As a
result, the general nature of the genetic mate
rial is that it is always nucleic acid; specifically,
it is DNA, except in the RNA viruses.
Ill Polynucleotide Chains
Have Nitrogenous Bases
Linked to a Sugar
Phosphate Backbone
Key concepts
• A nucleoside consists ofa purine or pyrimidine
base linked to the 1' carbon of a pentose sugar_
• The difference between DNA and RNA is in the group
at the 2' position ofthe sugar. DNA has a deoxyri
bose sugar {2'-H); RNA has a ribose sugar {2'-0H).
• A nucleotide consists of a nucleoside linked to a
phosphate group on either the S ' or 3' carbon of
the (deoxy)ribose.
• Successive (deoxy)ribose residues of a
polynucleotide chain are joined by a phosphate
group between the 3' carbon of one sugar and the
S' carbon of the next sugar.
• One end of the chain (conventionally written on
the left) has a free S' end and the other end of
the chain has a free 3' end.
• DNA contains the four bases adenine, guanine, cyto
sine, and thymine; RNA has uracil instead ofthymine.
The basic building block of nucleic adds (DNA
and RNA) is the nucleotide, which has three
components:
I. A nitrogenous base
2. A sugar
3. One or more phosphates
The nitrogenous base is a purine or pyrimi
dine ring. The base is linked to the 1 ' ("one
prime") carbon on a penrose sugar by a glyco
sidicbond from the N1 of pyrimidines or the N9
of purines. The penrose sugar linked to a nitro
genous base is called a nucleoside. To avoid
ambiguity between the numbering systems of
the heterocyclic rings and the sugar, positions
on the penrose are given a prime (').
Nucleic acids are named for the type of
sugar: DNA has 2'-deoxyribose, whereas RNA
has ribose. The difference is that the sugar in
RNA has a hydroxyl (-OH) group on the 2'
carbon of the penrose ring. The sugar can be
linked by its 5' or 3' carbon to a phosphate
group. A nucleoside linked to a phosphate is a
nucleotide.
Apolynucleotideis a longchain ofnucleo
tides. FIGURE 1.8 shows that the backbone of the
polynucleotide chain consists of an alternating
series of pentose (sugar) and phosphate resi
dues. The chain is formed by linking the 5' car
bon of one pentose ring to the 3' carbon of the
next pentose ring via a phosphate group; thus
the sugar-phosphate backbone is said to consist
of 5'-3' phosphodiester linkages. Specifically,
the 3' carbon of one penrose is bonded to one
oxygen of the phosphate, while the 5' carbon
of the other penrose is bonded to the opposite
oxygen of the phosphate. The nitrogenousbases
#stick outN from the backbone.
Each nucleic acid contains four types of
nitrogenous bases. The same two purines,
adenine (A) and guanine (G), are present in
both DNA and RNA. The two pyrimidines in
DNA are cytosine (C) and thymine (T); in RNA
Sugar-phosphate- H
backbone 2
5'-3' phospho
diester bonds
!---Nucleotide subunit
Pyrimidine base
Purine base
0
3,
FIGURE 1.8 A polynucleotide chain consists of a series of
5'-3' sugar-phosphate links that form a backbone from
which the bases protrude.
1.4 Polynucleotide Chains Have Nitrogenous Bases linked to a Sugar-Phosphate Backbone 7
36. uracil (U) is found instead ofthymine. The only
difference between uracil and thymine is the
presence of a methyl group at position c5.
The terminal nucleotide at one end of the
chain has a free 5' phosphate group, whereas
the terminal nucleotide at the other end has
a free 3' hydroxyl group. It is conventional
to write nucleic acid sequences in the 5' to 3'
direction-that is, from the 5' terminus at the
left to the 3' terminus at the right.
Ill Supercoiling Affects
the Structure of DNA
Key concepts
• Supercoiling occurs only in "closed" DNA with no
free ends.
• Closed DNA is either circular DNA or linear DNA in
which the ends are anchored so that they are not
free to rotate.
• A closed DNA molecule has a linking number
(
L
)
,
which is the sum oftwist (T) and writhe (W).
• The linking number can be changed only by breaking
and reforming bonds in the DNA backbone.
The two strands of DNA are wound around
each other to form a double helical structure
(described in detail in the next section); the
double helix can also wind around itself to
change the overall conformation, or topology,
of the DNA molecule in space. This is called
supercoiling. The effect can be imagined like a
rubberband twisted around itself. Supercoiling
creates tension in the DNA, and thus can only
occur if the DNA has no free ends (otherwise
the free ends can rotate to relieve the ten·
sion) or in linear DNA (FIGURE 1.9, top) if it is
anchored to a protein scaffold, as in eukaryotic
chromosomes. The simplest example of a DNA
with no free ends is a circular molecule. The
effect of supercoilingcan be seen by comparing
the nonsupercoiled circular DNA lying flat in
Figure 1.9 (center) with the supercoiled circular
molecule that forms a twisted (and therefore
more condensed) shape (Figure l.9, bottom).
The consequences of supercoiling depend
on whether the DNA is twisted around itself in
the same direction as the two strands within
the double helix (clockwise) or in the opposite
direction. Twisting in the same direction pro·
ducespositivesupercoiling, which overwinds the
DNA so that there are fewerbase pairs per turn.
Twisting in the opposite direction produces
negative supercoiling, or underwinding, so there
are more base pairs per turn. Both types of
supercoiling of the double helix in space are
8 CHAPTER 1 Genes Are DNA
FIGURE 1.9 Linear DNA is extended (top); a circular
DNA remains extended if it is relaxed (nonsupercoiled;
center); but a supercoiled DNA has a twisted and con
densed form (bottom). Photos courtesy of Nirupam Roy
Choudhury, International Centre for Genetic Engineering
and Biotechnology (ICGEB).
tensions in the DNA (which is why DNA mole
culeswith no supercoilingare called "relaxed").
Negative supercoiling can be thought of as
creating tension in the DNA that is relieved by
the unwinding of the double helix. The effect
of severe negative supercoiling is to generate a
region in which the two strands of DNA have
separated (technically, zerobase pairs per turn).
Topological manipulation of DNA is a cen
tral aspect of all its functional activities (recom
bination, replication, and transcription) as well
as of the organization of its higher order struc
ture. All synthetic activities involving double
stranded DNA require the strands to separate.
The strands do not simply lie sideby side though;
they are intertwined. Their separation therefore
requires the strands to rotate about each other
in space. Some possibilities for the unwinding
reaction are illustrated in FIGURE 1.10.
Unwinding a short linear DNA presents
no problems, as the DNA ends are free to spin
around the axis of the double helix to relieve
any tension. DNA in a typical chromosome,
however, is not only extremely long, but is also
coated with proteins that serve to anchor the
DNA at numerous points. As a result, even a
linear eukaryotic chromosome does not func
tionally possess free ends.
37. Rotation about a free end
Rotation at fixed ends
Strand separation compensated
by positive supercoiling
Nicking, rotation, and ligation
Nick
�
FIGURE 1.10 Separation ofthe strands of a DNA double
helix can be achieved in several ways.
Consider the effects of separating the two
strands in a molecule whose ends are not free
to rotate. When two intertwined strands are
pulled apart from one end, the result is to
iucrease their winding about each other farther
alongthe molecule, resultingin positive super
coiling elsewhere in the molecule tobalance the
underwindinggenerated in the single-stranded
region. Theproblemcanbe overcome by intro
ducing a transient nick in one strand. An inter
nal free end allows the nicked strand to rotate
about theintact strand, afterwhich the nick can
be sealed. Each repetition of the nicking and
sealing reaction releases one superhelical turn.
A closed molecule of DNA can be charac
terized by its linking number (L), which is
the number of times one strand crosses over
the other in space. Closed DNA molecules of
identical sequence may have different linking
numbers, reflecting different degrees of super
coiling. Molecules of DNA that are the same
except for their linking numbers are called
topological isomers.
The linkingnumberis made up oftwo com
ponents: the writhing number (W) and the
twisting number (T). The twisting number,
T, is a property of the double helical structure
itself, representing the rotation of one strand
about the other. It represents the total number
of turns of the duplex and is determined by the
number of base pairs per turn. For a relaxed
closed circular DNA lying flat in a plane, the
twistis the total munber ofbase pairs dividedby
the number ofbase pairsper tmn. The writhing
number, W, represents the turning of the axis
of the duplex in space. It corresponds to the
intuitive concept of supercoiling, but does not
have exactly the same quantitative definition or
measurement. For a relaxed molecule, W = 0,
and the linking number equals the twist.
We are often concerned with the change
in linking nwnber, �L. given by the equation:
�L = �W + �T
The equation states that any change in the total
ntm1ber ofrevolutions ofoneDNA strand about
the other can be expressed as the sum of the
changes of the coiling of the duplex axis in
space (�W) and changes in the helical repeat
of the double helix itself (�T}. In the absence of
proteinbinding or other constraints, the twist of
DNA doesnot tend to vary-in otherwords, the
10.5 base pairsperturn (bp/nnn) helical repeat
is a very stable conformation for DNA in solu
tion. Thus, any M (change in linking number)
is mostly likely to be expressed by a change in
W; that is, by a change in supercoiling.
A decrease in linking number (that is, a
change of-�L) corresponds to the introduction
of some combination of negative supercoiling
(�W) and/or underwinding (�T}. An increase
in linking number, measured as a change of
+�L, corresponds to an increase in positive
supercoiling and/or overwinding.
We can describe the change in state of
any DNA by the specific linking difference,
a = &/LO, for which LO is the linking number
when the DNA is relaxed. Ifall ofthe change in
linking number is due to change in W (that is,
�T = 0), the specific linking difference equals
the supercoiling density. In effect, a as defined
in terms ofMILO canbe assumedto correspond
to supercoiling density so long as the strucntre
of the double helix itself remains constant.
The critical feature about the use of the
linkingnumberisthatthispanuneteris an invari
ant property of any individual closed DNA mol
ecule. The linking number cannot be changed
by any defonnation short of one that involves
the breaking and rejoiningof strands. A circular
molecule with a particular linking number can
express the number in terms of different com
binations ofT and W, but it cannot change their
1.5 Supercoiling Affects the Structure of DNA 9