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1. 1. Lewin's GENES XI
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3. 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
4. 4. World Headquarters Jones & Bartlett Learning 5 Wall Street Burlington, MA 0 1 803 978-443-5000 info@jblearning.com www.jblearning.com Jones & Bartlett Learning books and products are available through most bookstores and online booksellers. To contact Jones & Bartlell Learning directly, call800-832-0034, fax 978- 443-8000, or visit our website, www.jblearning.com. Substantial discounts on bulk quantities of Jones & Bartlell Learning publications are available to corporations, professional associations, and other qualified organizations. For details and specific discount information, contact the special sales department at Jones & Bartlett Learning via the above contact information or send an email to specialsales@jblearning.com. Copyright© 2014 by Jones & B artlett Learning, LLC, an Ascend Learning Company All rights reserved. No part of the material protected by this copyright may be reproduced or utilized in any form, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without written permission from the copyright owner. Lewi11's GENES XI is an independent publication and has not been authorized, sponsored, or otherwise approved by the owners of the trademarks or service marks referenced in this product. Some images in this book feature models. These models do not necessarily endorse, represent, or participate in the activities represented in the images. Production Credits Chief Executive Officer: Ty Field President: James Homer SVP, Editor-in-Chief: Michael Johnson SVP, Chief Marketing Officer: Alison M . Pendergast Executive Publisher: Kevin Sullivan Senior Acquisitions Editor: Erin O'Connor Editorial Assistant: Rachel Isaacs Editorial Assistant: Michelle Bradbury Production Editor: Wendy Swanson Senior Marketing Manager: Andrea DeFronzo V.P., Manufacturing and Inventory Control: Therese Connell Composition: Circle Graphics, Inc. Cover Design: Kristin E. Parker Rights & Photo Research Associate: Lauren Miller Cover Image: © Kenneth EwardiScience Photo Library Printing and Binding: Courier Companies Cover Printing: Courier Companies To order this product, use ISBN: 978-1-4496-5985-1 Library of Congress Cataloging-in-Publication Data Krebs, Jocelyn E. Lewin's genes Xl. - 1 1th ed. I Jocelyn Krebs, Stephen Kilpatrick, and Elliott Goldstein. p.; em. Genes XI Lewin's genes I I Lewin's genes eleven Rev. ed. of: Lewin's genes X. 1Oth ed. I [edited by] Jocelyn E . Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick. c2011. Includes bibliographical references and index. ISBN 978-1-4496-590 5-9 (alk. paper) I. Lewin, Benjamin. II. Kilpatrick, Stephen T. Ill. Goldstein, Elliott S . IV. Lewin's genes X . V. Title. VI. Title: Genes XI. Vll. Title: Lewin's genes I I . VIII. Title: Lewin's genes eleven. [DNLM: I . Genes. 2. DNA-genetics. 3. Genetic Processes. 4. Genome. 5 . Proteins-genetics. 6. RNA-genetics. QU 470] 576.5-dc23 2 0 1 2020620 6048 Printed in the United States of America 1 7 16 1 5 1 4 1 3 1 0 9 8 7 6 5 4 3 2 I
5. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.
25. 25. Chapter Opener Credits and Captions Part 1 Photo courtesy of S. V. Flores, A. Mena, and B. F. McAllister. Used with pennission of Bryant McAllis­ ter, Department of Biology, University of Iowa. Chapter 1 A strand of DNA in blue and red. DNA is the genetic material of eukaryotic cells, bacteria, and many vimses. © Artsilensecome/ShutterStock, Inc. Chapter 2 A visual representation of gene expression analysis that allows researchers to see patterns in a network of expressed genes. Each dot represents a gene, and links between the dots occur where there are similar patterns of expression between the genes. © Anton Enright, The Sanger Institute/Wellcome Images. Chapter 3 DNA fragments separated by gel electrophoresis. © Nicemonkey/Dreamstime.com. Chapter 4 A self-splicing inn·on in an rRNA of the large ribosomal subunit. © Kenneth Eward/Photo Researchers, Inc. Chapter 5 Scanning electron micrograph (SEM) of human X and Y chromosomes in metaphase (35,000x). Most of the content of the genome is found in chromosomal DNA, though some organelles contain their own genomes. © Biophoto Associates/ Photo Researchers, Inc. Chapter 6 Mouse and human chromosomes. Both species' genomes have undergone chromosomal rearrange­ ments from the genome of their most recent common ancestor. The colors and corresponding numbers on the mouse chromosomes indicate the human chro­ mosomes containing homologous segments. Photo courtesy of U.S. Department of Energy. Used with permission of Lisa J. Stubbs, University of Illinois at Urbana-Champaign. Chapter 7 Scanning electron micrograph (SEM) of red blood cells. Genes that encode the protein subunits of hemoglobin, the oxygen-carrying complex found in red blood cells, are members of a large gene family that have evolved by duplication and divergence. © Sebastian Kaulitzki/ ShutterStock. Inc. Chapter S A circular genome map showing shared (syntenic) regions between humans (outer ring) and (from inner ring outward) chimpanzee, mouse, rat, dog, chicken, and zebrafish genomes. Similar colors among rings show synteny. © Martin Krzywinski/Photo Research­ ers, Inc. Chapter 9 Microphotograph of living cells undergoing mitosis. Individual chromosomes lined up at the metaphase plate or separating are visible in several cells. © Dimar­ ion/ShutterStock, Inc. Chapter 10 A three-dimensional model of chromatin showing one possible arrangement for the nucleosomes in the 30-nm fiber. Photo courtesy of Thomas Bishop, Tulane University, using VDNA plug-in for VMD (http: IIwww. ks. ui uc. edu/research/vmd/plugins/ vdna/ ). Part 2 Image courtesy of Teresa Larsen, The Foundation for Scientific Literacy. Chapter 1 1 A transmission electron micrograph of an E. coli bac­ terium in the mid to late stages of binary fission, the process by which the bacterium divides. The hair­ like appendages around the bacterium are pili, stmc­ tures used for bacterial conjugation (magnification: l7,500x). © CNRI/Photo Researchers, Inc. XXV
26. 26. Chapter 12 Computer model of DNA polymerase replicating a strand of DNA (across center). The secondary struc­ ture of the DNA polymerase and the primary structure of the DNA molecule are shown. DNA polymerases are enzymes that synthesize strands of DNA from a complementary template strand during DNA replica­ tion. This molecule is in the Y family of DNA polymer­ ases, which an: translesion synthesis polymerases, that is, they are able to replicate damaged areas of DNA that stall other DNA polymerases. The replication is not always accurate and can lead to mutagenesis or cancer. © Laguna Design/Photo Researchers, Inc. Chapter 13 A type II topoisomerase acting on a knotted or super­ coiled DNA substrate. The cleaved DNA is shown in green. The DNA that is being passed through the break is viewed end-on. Photo courtesy of James Berger, University of California, Berkeley. Chapter 14 Plasmids. © Deco Images II/Almay Images. Chapter 15 A model demonstrating the interaction of a RuvA ret­ ramer with a DNA Holliday junction, an intermedi­ ate of recombination. Reproduced with permission from J. B. Rafferty et a!., Science 274 ( 1996): 41 5-421 . © 1996 AAAS. Photo courtesy of David W. Rice and John B. Rafferty, University of Sheffield. Chapter 1 6 The XPD helicase, involved in both transcription and nucleotide excision repair, is shown unwinding DNA. Colors indicate different domains of XPD; the heli­ case domain is green. Photo courtesy of M. Pique, Li Fan, Jill Fuss, and John Tainer, The Scripps Research Institute and Lawrence Berkeley National Laboratory. More information available at L. Fan et a!., Cell 1 33 (2008): 789-800. Chapter 1 7 Artist's rendering of an electron micrograph showing retroviruses. © MedicalR F/Photo Researchers, Inc. Chapter 18 Human lg autoantibody binding double-strand DNA. Rendered by UCSF Chimera, P. Casali & E.J. Pone, 2012. Part 3 Photo courtesy of Jie Ren and Jun Ma, University of Cincinnati College of Medicine and Cincinnati Chil­ dren's Hospital. xxvi Chapter Opener Credits and Captions Chapter 19 Transcription factor and DNA molecules. Molecular model showing the secondary structure (helices) of the transcription factor Brachyury (purple) binding to a molecule of DNA (deoxyribonucleic acid, run­ rung horizontally from left to right). Brachyury, also known as T, is a protein that controls the transcrip­ tion of DNA to RNA (ribonucleic acid) by the enzyme RNA polymerase. RNA is the intermediate product in the fonnation of a protein from its gene. Brachyury binds to a specific sequence of bases in the DNA called a T-box. Brachyury has been found to be crucial in embryonic development in animals. It is necessary to fom1 the mesoderm, the precursor tissue that even­ tually develops into the internal organs and bones. © Phantatomix/Photo Researchers, Inc. Chapter 20 Biomedical illustration of transcription showing RNA polymerase at work. A DNA sequence is copied by RNA polymerase to produce a complementary nucle­ otide RNA strand, called messenger RNA (mRNA). © Carol & Mike Werner/Visuals Unlimited. Chapter 21 A three-dimensional structure of the mammalian spliceosomal C complex. Spliceosome structure cour­ tesy of Nikolaus Grigorieff, Brandeis University. Back­ ground photo © Bella D/ShutterStock, Inc. Chapter22 Computer artwork of aconitase (blue), in complex with ferritin mRNA (red). Aconitase is involved in the citric acid cycle, but here it is performing a second­ ary function as an iron regulatory protein (IRP). It does this by binding to ferritin mRNA, which prevents translation into the protein product (ferritin). Ferritin acts like a sponge and helps to protect cells from the toxic effects of excess iron. © Equinox Graphics/Photo Researchers, Inc. Chapter 23 The figure shows the RNA/protein architecture of the large ribosomal subunit with the active site high­ lighted. The background shows a schematic diagram of the peptidyl transferase active site of the ribosome. Photo courtesy of Nenad Ban, Institute for Molecular Biology & Biophysics, Zurich. Chapter 24 Reproduced from I. L. Mainprize, et a!., MolecularBiol­ OfJY of the Cell 17 (2006): 5063-5074. Copyright 2006 by American Society of Cell Biology. Used with per­ mission of Courtney Hodges, University of California,
27. 27. Berkeley, and Laura Lancaster, University of California, Santa Cruz. Photo courtesy of Carlos Bustamante, University of California, Berkeley. Chapter25 An aminoacyl-tRNA synthetase is shown complexed to a tRNA. Reproduced from M. Blaise et al., Nucleic Acids Res. 32, (2004): 2768-2775. Used with permis­ sion of Oxford University Press. Photo courtesy of Pr. Daniel Kern, Universite Louis Pasteur, Imtitut de Biologie Moleculaire et Cellulaire, UPR 9002 du SNRS, Strasbourg. Part 4 © 2007, Sangamo BioSciences, Inc. (www.sangamo. com). Chapter 26 A visualization showing the structure of the lac repressor protein as it binds with DNA. Figure created by Elizabeth Villa using VMD and under copyright of the Theoretical and Computational Biophysics Group, NIH Resource for Macromolecular Modeling and Bioinfom1atics, at the Beckman Institute, Uni­ versity of Illinois at Urbana-Champaign. The work was partially supported by the National Center for Supercomputing Applications under National Insti­ tutes of Health Grant PHS5P41 RR05969-04 and National Science Foundation Grant MCB-9982629. Computer time was provided by National Resource Allocations Committee Grant MCA93S028. Chapter 27 A transmission electron micrograph of a T4 phage (magnification: 450,000,000x). © Dr. Harold Fisher/ Visuals Unlimited. Chapter28 A model of the ATP-dependent chromatin-remodeling complex RSC bound to a nucleosome. Photo courtesy of Andres Leschziner, Harvard University. Chapter29 Structural model for the cohesin complex consist­ ing of Smcl (red), Smc3 (blue), and Sccl (green) proteins encircling two sister chromatids, and DNA (gold) wrapped around nucleosomes (gray). Photo courtesy of Christian Haering, European Molecular Biology Laboratory-Germany. Used with permis­ sion of Kim Nasmyth, University of Oxford-United Kingdom. Chapter 30 A hammerhead ribozyme that catalyzes a self-cleaving reaction. Many plant viroids are han1merhead RNAs. Reproduced from M. Martick et al., Solvent Structure and Hammerhead Ribozyme Catalysis. Chern. Bio. 1 5, pp. 332-342. Copy1ight 2008, with permission from Elsevier (hup://www.sciencedirect.com/science/ journal/ 10745521 ). Photo courtesy of William Scott, University of California, Santa Cruz. Chapter Opener Credits and Captions xxvii
28. 28. 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
29. 29. 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.
30. 30. 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
31. 31. 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.
32. 32. 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
33. 33. 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.
34. 34. 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
35. 35. 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.
36. 36. 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

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