মুখ্য Molecular Biology of the Gene
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MOLECULAR BIOLOGY GENE OF THE S E V E N T H E D I T I O N This page intentionally left blank MOLECULAR BIOLOGY GENE OF THE S E V E N T H E D I T I O N J AMES D. WATSON A LEXANDER G ANN Cold Spring Harbor Laboratory Cold Spring Harbor Laboratory TANIA A. B AKER M ICHAEL L EVINE Massachusetts Institute of Technology University of California, Berkeley S TEPHEN P. B ELL R ICHARD LOSICK Massachusetts Institute of Technology Harvard University With S TEPHEN C. H ARRISON Harvard Medical School (Chapter 6: The Structure of Proteins) Boston Columbus Indianapolis New York San Francisco Upper Saddle River Amsterdam Cape Town Dubai London Madrid Milan Munich Paris Montréal Toronto Delhi Mexico City São Paulo Sydney Hong Kong Seoul Singapore Taipei Tokyo C O L D S P R I N G H A R B O R L A B O R AT O R Y P R E S S Cold Spring Harbor, New York PEARSON COLD SPRING HARBOR LABORATORY PRESS Publisher and Sponsoring Editor: John Inglis Editor-in-Chief: Beth Wilbur Editorial Director: Alexander Gann Senior Acquisitions Editor: Josh Frost Director of Editorial Development: Jan Argentine Executive Director of Development: Deborah Gale Managing Editor and Developmental Editor: Kaaren Janssen Assistant Editor: Katherine Harrison-Adcock Project Manager: Inez Sialiano Managing Editor: Michael Early Production Manager: Denise Weiss Production Project Manager: Lori Newman Production Editor: Kathleen Bubbeo Illustrators: Dragonfly Media Group Permissions Coordinator: Carol Brown Manufacturing Buyer: Michael Penne Crystal Structure Images: Leemor Joshua-Tor and Stephen C. Harrison Director of Marketing: Christy Lesko Cover Designer: Mike Albano Executive Marketing Manager: Lauren Harp Executive Media Producer: Laura Tommasi Editorial Media Producer: Lee Ann Doctor Supervising Media Project Manager: David Chavez Director of Content Development, MasteringBiology: Natania Mlawer Content Specialist, MasteringBiology: J. Zane Barlow, PhD Front and Back Cover Images: Far left, drawing by Francis Crick, Wellcome L; ibrary, London. Second from left, from Watson J.D. and Crick F.H.C. 1953. Nature 171: 737– 738. Second from right, Irving Geis illustration. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission. Far right, structure by Leemor Joshua-Tor (image prepared with PyMOL). Credits and acknowledgments for materials borrowed from other sources and reproduced, with permission, in this textbook appear on the appropriate page within the text. Copyright # 2014, 2008, 2004 Pearson Education, Inc. All rights reserved. Manufactured in the United States of America. This publication is protected by Copyright, and permission should be obtained from the publisher prior to any prohibited reproduction, storage in a retrieval system, or transmission in any form or by any means, electronic, mechanical, photocopying, recording, or likewise. To obtain permission(s) to use material from this work, please submit a written request to Pearson Education, Inc., Permissions Department, 1900 E. Lake Ave., Glenview, IL 60025. For information regarding permissions, call (847) 486-2635. Readers may view, browse, and/or download material for temporary copying purposes only, provided these uses are for noncommercial personal purposes. Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or in part, without prior written permission from the publisher. Many of the designations used by manufacturers and sellers to distinguish their products are claimed as trademarks. Where those designations appear in this book, and the publisher was aware of a trademark claim, the designations have been printed in initial caps or all caps. MasteringBiology and BioFlix are trademarks, in the U.S. and/or other countries, of Pearson Education, Inc. or its affiliates. Library of Congress Cataloging-in-Publication Data Watson, James D. Molecular biology of the gene / James D. Watson, Cold Spring Harbor Laboratory, Tania A. Baker, Massachusetts Institute of Technology, Alexander Gann, Cold Spring Harbor Laboratory, Michael Levine, University of California, Berkeley, Richard Losick, Harvard University. pages cm Includes bibliographical references and index. ISBN-13: 978-0-321-76243-6 (hardcover (student ed)) ISBN-10: 0-321-76243-6 (hardcover (student ed)) ISBN-13: 978-0-321-90537-6 ( paper (a la carte)) ISBN-10: 0-321-90537-7 ( paper (a la carte)) [etc.] 1. Molecular biology- -Textbooks. 2. Molecular genetics- -Textbooks. I. Title. QH506.M6627 2013 572’.33--dc23 2012046093 1 2 3 4 5 6 7 8 9 10—DOW—17 16 15 14 13 www.pearsonhighered.com COLD SPRING HARBOR LABORATORY PRESS www.cshlpress.org ISBN 10: 0-321-76243-6 (Student Edition) ISBN 13: 978-0-321-76243-6 (Student Edition) ISBN 10: 0-321-90264-5 (Instructor’s Review Copy) ISBN 13: 978-0-321-90264-1 (Instructor’s Review Copy) ISBN 10: 0-321-90537-7 (Books à la Carte Edition) ISBN 13: 978-0-321-90537-6 (Books à la Carte Edition) Preface T MOLECULAR BIOLOGY OF THE GENE appears in this, its 7th edition, on the 60th anniversary of the discovery of the structure of DNA in 1953, an occasion celebrated by our cover design. The double-helical structure, held together by specific pairing between the bases on the two strands, has become one of the iconic images of science. The image of the microscope was perhaps the icon of science in the late 19th century, displaced by the mid 20th century by the graphical representation of the atom with its orbiting electrons. But by the end of the century that image had in turn given way to the double helix. The field of molecular biology as we understand it today was born out of the discovery of the DNA structure and the agenda for research that that structure immediately provided. The paper by Watson and Crick proposing the double helix ended with a now famous sentence: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” The structure suggested how DNA could replicate, opening the way to investigate, in molecular terms, how genes are passed down through generations. It was also immediately apparent that the order of bases along a DNA molecule could represent a “genetic code,” and so an attack on that second great mystery of genetics—how genes encode characteristics—could also be launched. By the time the first edition of Molecular Biology of the Gene was published, just 12 years later in 1965, it had been confirmed that DNA replicated in the manner suggested by the model, the genetic code had all but been cracked, and the mechanism by which genes are expressed, and how that expression is regulated, had been established at least in outline. The field of molecular biology was ripe for its first textbook, defining for the first time the curriculum for undergraduate courses in this topic. Our understanding of the mechanisms underlying these processes has hugely increased over the last 48 years since that first edition, often driven by technological advances, including DNA sequencing (another anniversary this year is the 10th anniversary of completion of the human genome project). The current edition of Molecular Biology of the Gene celebrates both the central intellectual framework of the field put in place in that first edition and the extraordinary mechanistic, biological, and evolutionary understanding that has since been achieved. HE NEW EDITION OF New to This Edition There are a number of major changes to the new edition. As well as wide-ranging updates, these include changes in organization, addition of completely new chapters, and the addition of new topics within existing chapters. . New Part 2 on the Structure and Study of Macromolecules. In this new section, each of the three major macromolecules gets its own chapter. The DNA chapter is retained from the previous edition, but what was previously just a short section at the end of that chapter is now expanded into a whole new chapter on the structure of RNA. The chapter on the structure of proteins is completely new and was written for this edition by Stephen Harrison (Harvard University). v vi Preface . Techniques chapter moved from the end of the book into Part 2. This revised and relocated chapter introduces the important techniques that will be referred to throughout the book. In addition to many of the basic techniques of molecular biology, this chapter now includes an updated section on many genomics techniques routinely employed by molecular biologists. Techniques more specialized for particular chapters appear as boxes within those chapters. . Completely new chapter on The Origin and Early Evolution of Life. This chapter shows how the techniques of molecular biology and biochemistry allow us to consider—even reconstruct—how life might have arisen and addresses the prospect of creating life in a test tube (synthetic biology). The chapter also reveals how, even at the very early stages of life, molecular processes were subject to evolution. . New material on many aspects of gene regulation. Part 5 of the book is concerned with gene regulation. In this edition we have introduced significant new topics, such as quorum sensing in bacterial populations, the bacterial CRISPR defense system and piRNAs in animals, the function of Polycomb, and increased discussion of other so-called “epigenetic” mechanisms of gene regulation in higher eukaryotes. The regulation of “paused polymerase” at many genes during animal development and the critical involvement of nucleosome positioning and remodeling at promoters during gene activation are also new topics to this edition. . End-of-chapter questions. Appearing for the first time in this edition, these include both short answer and data analysis questions. The answers to the even-numbered questions are included as Appendix 2 at the back of the book. . New experiments and experimental approaches reflecting recent advances in research. Integrated within the text are new experimental approaches and applications that broaden the horizons of research. These include, for example, a description of how the genetic code can be experimentally expanded to generate novel proteins, creation of a synthetic genome to identify the minimal features required for life, discussion of new genomewide analysis of nucleosome positioning, experiments on bimodal switches in bacteria, and how new antibacterial drugs are being designed that target the quorum-sensing pathways required for pathogenesis. Supplements MasteringBiology www.masteringbiology.com MasteringBiology is an online homework, tutorial, and assessment system that delivers self-paced tutorials that provide individualized coaching, focus on your course objectives, and are responsive to each student’s progress. The Mastering system helps instructors maximize class time with customizable, easy-to-assign, and automatically graded assessments that motivate students to learn outside of class and arrive prepared for lecture. MasteringBiology includes the book’s end-of-chapter problems, eighteen 3D structure tutorials, reading quizzes, animations, videos, and a wide variety of activities. The eText is also available through MasteringBiology, providing access to the complete textbook and featuring powerful interactive and customization functions. Instructor Resource DVD 978-0-321-88342-1/0-321-88342-X Available free to all adopters, this dual-platform DVD-ROM contains all art and tables from the book in JPEG and PowerPoint in high-resolution (150 dpi) files. The PowerPoint slides include problems formatted for use with Classroom Response Systems. This DVDROM also contains an answer key for all of the end-of-chapter Critical Thinking questions included in MasteringBiology. Transparency Acetates 978-0-321-88341-4/0-321-88341-1 Features approximately 90 four-color illustrations from the text. These transparencies are free to all adopters. Preface Cold Spring Harbor Laboratory Photographs As in the previous edition, each part opener includes photographs, some newly added to this edition. These pictures, selected from the archives of Cold Spring Harbor Laboratory, were all taken at the Lab, the great majority during the Symposia hosted there almost every summer since 1933. Captions identify who is in each picture and when it was taken. Many more examples of these historic photos can be found at the CSHL archives website (http:// archives.cshl.edu/). Acknowledgments Parts of the current edition grew out of an introductory course on molecular biology taught by one of us (RL) at Harvard University, and this author is grateful to Steve Harrison and Jim Wang who contributed to this course in past years. In the case of Steve Harrison, we are additionally indebted to him for writing and illustrating a brand new chapter on protein structure especially for this new edition. No one could be better qualified for such a task, and we are the grateful beneficiaries of—and the book is immeasurably improved by—his contribution. We are also grateful to Craig Hunter, who earlier wrote the section on the worm for Appendix 1, and to Rob Martienssen, who wrote the section on plants for that same appendix. We have shown sections of the manuscript to various colleagues and their comments have been extremely helpful. Specifically we thank Katsura Asano, Stephen Blacklow, Jamie Cate, Amy Caudy, Irene Chen, Victoria D’Souza, Richard Ebright, Mike Eisen, Chris Fromme, Brenton Graveley, Chris Hammell, Steve Hahn, Oliver Hobert, Ann Hochschild, Jim Hu, David Jerulzalmi, Leemor Joshua-Tor, Sandy Johnson, Andrew Knoll, Adrian Krainer, Julian Lewis, Sue Lovett, Karolin Luger, Kristen Lynch, Rob Martienssen, Bill McGinnis, Matt Michael, Lily Mirels, Nipam Patel, Mark Ptashne, Danny Reinberg, Dimitar Sasselov, David Shechner, Sarah T. Stewart-Mukhopadhyay, Bruce Stillman, and Jack Szostak. We also thank those who provided us with figures, or the wherewithal to create them: Sean Carroll, Seth Darst, Paul Fransz, Brenton Graveley, Ann Hochschild, Julian Lewis, Bill McGinnis, Phoebe Rice, Dan Rokhsar, Nori Satoh, Matt Scott, Ali Shilatifard, Peter Sorger, Tom Steitz, Andrzej Stasiak, Dan Voytas, and Steve West. New to this edition are end-of-chapter questions, provided by Mary Ellen Wiltrout, and we thank her for these efforts that have enhanced the new edition. In addition, Mary Ellen helped with revisions to the DNA repair chapter. We are indebted to Leemor Joshua-Tor, who so beautifully rendered the majority of the structure figures throughout the book. Her skill and patience are much appreciated. We are also grateful to those who provided their software1: Per Kraulis, Robert Esnouf, Ethan Merritt, Barry Honig, and Warren Delano. Coordinates were obtained from the Protein Data Bank (www.rcsb.org/pdb/), and citations to those who solved each structure are included in the figure legends. Our art program was again executed by a team from the Dragonfly Media Group, led by Craig Durant. Denise Weiss and Mike Albano produced a beautiful cover design. We thank Clare Bunce and the CSHL Archive for providing the photos for the part openers and for much help tracking them down. We thank Josh Frost at Pearson who oversaw our efforts and was always on hand to help us out or provide advice. In development at CSHL Press, Jan Argentine provided great support, guidance, and perspective throughout the process. Our heartfelt thanks to Kaaren Janssen who was once again our constant savior—editing and organizing, encouraging and understanding—and unstintingly good-humored even on the darkest days. Inez Sialiano kept track of the output, and Carol Brown dealt with the permissions as efficiently as ever. In production, we relied heavily on the extraordinary efforts and patience vii viii Preface of Kathleen Bubbeo, for which we are most grateful. And we must also thank Denise Weiss, who oversaw production and ensured that the book looked so good by finessing the page layout and creating the design. John Inglis as ever created the environment in which this could all take place. And once again, we thank our families for putting up with this book for a third time! JAMES D. WATSON TANIA A. BAKER STEPHEN P. BELL ALEXANDER GANN MICHAEL LEVINE RICHARD LOSICK 1 Per Kraulis granted permission to use MolScript (Kraulis P.J. 1991. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst. 24: 946–950). Robert Esnouf gave permission to use BobScript (Esnouf R.M. 1997. J. Mol. Graph. 15: 132–134). In addition, Ethan Merritt gave us use of Raster3D (Merritt E.A. and Bacon D.J. 1997. Raster3D: Photorealistic molecular graphics. Methods Enzymol. 277: 505– 524), and Barry Honig granted permission to use GRASP (Nicolls A., Sharp K.A., and Honig B. 1991. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11: 281– 296). Warren DeLano agreed to the use of PyMOL (DeLano W.L. 2002. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, California). About the Authors JAMES D. WATSON is Chancellor Emeritus at Cold Spring Harbor Laboratory, where he was previously its Director from 1968 to 1993, President from 1994 to 2003, and Chancellor from 2003 to 2007. He spent his undergraduate years at the University of Chicago and received his Ph.D. in 1950 from Indiana University. Between 1950 and 1953, he did postdoctoral research in Copenhagen and Cambridge, England. While at Cambridge, he began the collaboration that resulted in the elucidation of the double-helical structure of DNA in 1953. (For this discovery, Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in 1962.) Later in 1953, he went to the California Institute of Technology. He moved to Harvard in 1955, where he taught and did research on RNA synthesis and protein synthesis until 1976. He was the first Director of the National Center for Genome Research of the National Institutes of Health from 1989 to 1992. Dr. Watson was sole author of the first, second, and third editions of Molecular Biology of the Gene, and a co-author of the fourth, fifth and sixth editions. These were published in 1965, 1970, 1976, 1987, 2003, and 2007, respectively. He is also a co-author of two other textbooks, Molecular Biology of the Cell and Recombinant DNA, as well as author of the celebrated 1968 memoir, The Double Helix, which in 2012 was listed by the Library of Congress as one of the 88 Books That Shaped America. TANIA A. BAKER is the Head of the Department and Whitehead Professor of Biology at the Massachusetts Institute of Technology and an Investigator of the Howard Hughes Medical Institute. She received a B.S. in biochemistry from the University of Wisconsin, Madison, and a Ph.D. in biochemistry from Stanford University in 1988. Her graduate research was carried out in the laboratory of Professor Arthur Kornberg and focused on mechanisms of initiation of DNA replication. She did postdoctoral research in the laboratory of Dr. Kiyoshi Mizuuchi at the National Institutes of Health, studying the mechanism and regulation of DNA transposition. Her current research explores mechanisms and regulation of genetic recombination, enzyme-catalyzed protein unfolding, and ATP-dependent protein degradation. Professor Baker received the 2001 Eli Lilly Research Award from the American Society of Microbiology and the 2000 MIT School of Science Teaching Prize for Undergraduate Education and is a Fellow of the American Academy of Arts and Sciences since 2004 and was elected to the National Academy of Sciences in 2007. She is co-author (with Arthur Kornberg) of the book DNA Replication, Second Edition. STEPHEN P. BELL is a Professor of Biology at the Massachusetts Institute of Technology and an Investigator of the Howard Hughes Medical Institute. He received B.A. degrees from the Department of Biochemistry, Molecular Biology, and Cell Biology and the Integrated Sciences Program at Northwestern University and a Ph.D. in biochemistry at the University of California, Berkeley, in 1991. His graduate research was carried out in the laboratory of Dr. Robert Tjian and focused on eukaryotic transcription. He did postdoctoral research in the laboratory of Dr. Bruce Stillman at Cold Spring Harbor Laboratory, working on the initiation of eukaryotic DNA replication. His current research focuses on the mechanisms controlling the duplication of eukaryotic chromosomes. Professor Bell received the 2001 ASBMB – Schering Plough Scientific Achievement Award, the ix x About the Authors 1998 Everett Moore Baker Memorial Award for Excellence in Undergraduate Teaching at MIT, the 2006 MIT School of Science Teaching Award, and the 2009 National Academy of Sciences Molecular Biology Award. ALEXANDER GANN is the Lita Annenberg Hazen Dean and Professor in the Watson School of Biological Sciences at Cold Spring Harbor Laboratory. He is also a Senior Editor at Cold Spring Harbor Laboratory Press. He received his B.Sc. in microbiology from University College London and a Ph.D. in molecular biology from The University of Edinburgh in 1989. His graduate research was carried out in the laboratory of Noreen Murray and focused on DNA recognition by restriction enzymes. He did postdoctoral research in the laboratory of Mark Ptashne at Harvard, working on transcriptional regulation, and that of Jeremy Brockes at the Ludwig Institute of Cancer Research at University College London, where he worked on newt limb regeneration. He was a Lecturer at Lancaster University, United Kingdom, from 1996 to 1999, before moving to Cold Spring Harbor Laboratory. He is co-author (with Mark Ptashne) of the book Genes & Signals (2002) and co-editor (with Jan Witkowski) of The Annotated and Illustrated Double Helix (2012). MICHAEL LEVINE is a Professor of Genetics, Genomics and Development at the University of California, Berkeley, and is also Co-Director of the Center for Integrative Genomics. He received his B.A. from the Department of Genetics at the University of California, Berkeley, and his Ph.D. with Alan Garen in the Department of Molecular Biophysics and Biochemistry from Yale University in 1981. As a Postdoctoral Fellow with Walter Gehring and Gerry Rubin from 1982 to 1984, he studied the molecular genetics of Drosophila development. Professor Levine’s research group currently studies the gene networks responsible for the gastrulation of the Drosophila and Ciona (sea squirt) embryos. He holds the F. Williams Chair in Genetics and Development at University of California, Berkeley. He was awarded the Monsanto Prize in Molecular Biology from the National Academy of Sciences in 1996 and was elected to the American Academy of Arts and Sciences in 1996 and the National Academy of Sciences in 1998. RICHARD LOSICK is the Maria Moors Cabot Professor of Biology, a Harvard College Professor, and a Howard Hughes Medical Institute Professor in the Faculty of Arts and Sciences at Harvard University. He received his A.B. in chemistry at Princeton University and his Ph.D. in biochemistry at the Massachusetts Institute of Technology. Upon completion of his graduate work, Professor Losick was named a Junior Fellow of the Harvard Society of Fellows when he began his studies on RNA polymerase and the regulation of gene transcription in bacteria. Professor Losick is a past Chairman of the Departments of Cellular and Developmental Biology and Molecular and Cellular Biology at Harvard University. He received the Camille and Henry Dreyfus Teacher-Scholar Award and is a member of the National Academy of Sciences, a Fellow of the American Academy of Arts and Sciences, a Fellow of the American Association for the Advancement of Science, a Fellow of the American Academy of Microbiology, a member of the American Philosophical Society, and a former Visiting Scholar of the Phi Beta Kappa Society. Professor Losick is the 2007 winner of the Selman A. Waksman Award of the National Academy of Sciences, a 2009 winner of the Canada Gairdner Award, a 2012 winner of the Louisa Gross Horwitz Prize for Biology or Biochemistry of Columbia University, and a 2012 winner of the Harvard University Fannie Cox Award for Excellence in Science Teaching. Class Testers and Reviewers We wish to thank all of the instructors for their thoughtful suggestions and comments on versions of many chapters in this book. Chapter Reviewers Ann Aguanno, Marymount Manhattan College Robert B. Helling, University of Michigan David P. Aiello, Austin College David C. Higgs, University of Wisconsin, Parkside Charles F. Austerberry, Creighton University Mark Kainz, Colgate University David G. Bear, University of New Mexico Health Gregory M. Kelly, University of Western Ontario Sciences Center Margaret E. Beard, College of the Holy Cross Gail S. Begley, Northeastern University Sanford Bernstein, San Diego State University Michael Blaber, Florida State University Nicole Bournias, California State University, San Bernardino John Boyle, Mississippi State University Suzanne Bradshaw, University of Cincinnati John G. Burr, University of Texas at Dallas Michael A. Campbell, Pennsylvania State University, Erie, The Behrend College Ann Kleinschmidt, Allegheny College Dan Krane, Wright State University Mark Levinthal, Purdue University Gary J. Lindquester, Rhodes College James Lodolce, Loyola University Chicago Curtis Loer, University of San Diego Virginia McDonough, Hope College Michael J. McPherson, University of Leeds Victoria Meller, Tufts University William L. Miller, North Carolina State University Aaron Cassill, University of Texas at San Antonio Dragana Miskovic, University of Waterloo Shirley Coomber, King’s College, University of London David Mullin, Tulane University Anne Cordon, University of Toronto Jeffrey D. Newman, Lycoming College Sumana Datta, Texas A&M University James B. Olesen, Ball State University Jeff DeJong, University of Texas at Dallas Anthony J. Otsuka, Illinois State University Jurgen Denecke, University of Leeds Karen Palter, Temple University Susan M. DiBartolomeis, Millersville University James G. Patton, Vanderbilt University Santosh R. D’Mello, University of Texas at Dallas Ian R. Phillips, Queen Mary, University of London Robert J. Duronio, University of North Carolina, Chapel Hill Steve Picksley, University of Bradford Steven W. Edwards, University of Liverpool Debra Pires, University of California, Los Angeles David Frick, University of Wisconsin Todd P. Primm, University of Texas at El Paso Allen Gathman, Southeast Missouri State University Phillip E. Ryals, The University of West Florida Anthony D.M. Glass, University of British Columbia Eva Sapi, University of New Haven Elliott S. Goldstein, Arizona State University Jon B. Scales, Midwestern State University Ann Grens, Indiana University, South Bend Michael Schultze, University of York Gregory B. Hecht, Rowan University Venkat Sharma, University of West Florida xi xii Class Testers and Reviewers Erica L. Shelley, University of Toronto at Mississauga Class Testers Elizabeth A. Shephard, University College, London Charles F. Austerberry, Creighton University Margaret E. Stevens, Ripon College Christine E. Bezotté, Elmira College Akif Uzman, University of Houston, Downtown Astrid Helfant, Hamilton College Quinn Vega, Montclair State University Gerald Joyce, The Scripps Research Institute Jeffrey M. Voight, Albany College of Pharmacy Jocelyn Krebs, University of Alaska, Anchorage Lori L. Wallrath, University of Iowa Cran Lucas, Louisiana State University in Shreveport Robert Wiggers, Stephen F. Austin State University Anthony J. Otsuka, Illinois State University Bruce C. Wightman, Muhlenberg College Charles Polson, Florida Institute of Technology Bob Zimmermann, University of Massachusetts Ming-Che Shih, University of Iowa Brief Contents PART 1 PART 4 5' 3' A Aa A AA 3' aa Aa HISTORY, 1 5' 3' EXPRESSION OF THE GENOME, 423 1 The Mendelian View of the World, 5 13 Mechanisms of Transcription, 429 2 Nucleic Acids Convey Genetic Information, 21 14 RNA Splicing, 467 15 Translation, 509 PART 2 16 The Genetic Code, 573 17 The Origin and Early Evolution of Life, 593 STRUCTURE AND STUDY OF MACROMOLECULES, 45 PART 5 3 The Importance of Weak and Strong Chemical Bonds, 51 REGULATION, 609 4 The Structure of DNA, 77 5 The Structure and Versatility of RNA, 107 18 Transcriptional Regulation in Prokaryotes, 615 6 The Structure of Proteins, 121 19 Transcriptional Regulation in Eukaryotes, 657 7 Techniques of Molecular Biology, 147 20 Regulatory RNAs, 701 PART 3 21 Gene Regulation in Development and Evolution, 733 22 Systems Biology, 775 MAINTENANCE OF THE GENOME, 193 PART 6 8 Genome Structure, Chromatin, and the Nucleosome, 199 APPENDICES, 793 9 The Replication of DNA, 257 10 The Mutability and Repair of DNA, 313 11 Homologous Recombination at the Molecular Level, 341 12 Site-Specific Recombination and Transposition of DNA, 377 1 Model Organisms, 797 2 Answers, 831 Index, 845 xiii This page intentionally left blank Detailed Contents PART 1: HISTORY, 1 Aa AA aa Aa 1 The Mendelian View of the World, 5 MENDEL’S DISCOVERIES, 6 The Principle of Independent Segregation, 6 ADVANCED CONCEPTS BOX 1-1 Mendelian Laws, 6 Some Alleles Are neither Dominant nor Recessive, 7 Principle of Independent Assortment, 8 THE ORIGIN OF GENETIC VARIABILITY THROUGH MUTATIONS, 13 EARLY SPECULATIONS ABOUT WHAT GENES ARE AND HOW THEY ACT, 15 CHROMOSOMAL THEORY OF HEREDITY, 8 PRELIMINARY ATTEMPTS TO FIND A GENE– PROTEIN RELATIONSHIP, 16 GENE LINKAGE AND CROSSING OVER, 9 SUMMARY, 17 KEY EXPERIMENTS BOX 1-2 Genes Are Linked to Chromosomes, 10 CHROMOSOME MAPPING, 11 BIBLIOGRAPHY, 17 QUESTIONS, 18 2 Nucleic Acids Convey Genetic Information, 21 AVERY’S BOMBSHELL: DNA CAN CARRY GENETIC SPECIFICITY, 22 Viral Genes Are Also Nucleic Acids, 23 THE DOUBLE HELIX, 24 KEY EXPERIMENTS BOX 2-1 Chargaff’s Rules, 26 Finding the Polymerases That Make DNA, 26 Experimental Evidence Favors Strand Separation during DNA Replication, 27 THE GENETIC INFORMATION WITHIN DNA IS CONVEYED BY THE SEQUENCE OF ITS FOUR NUCLEOTIDE BUILDING BLOCKS, 30 KEY EXPERIMENTS BOX 2-2 Evidence That Genes Control Amino Acid Sequences in Proteins, 31 DNA Cannot Be the Template That Directly Orders Amino Acids during Protein Synthesis, 32 RNA Is Chemically Very Similar to DNA, 32 THE CENTRAL DOGMA, 33 The Adaptor Hypothesis of Crick, 34 Discovery of Transfer RNA, 34 The Paradox of the Nonspecific-Appearing Ribosomes, 35 Discovery of Messenger RNA (mRNA), 35 Enzymatic Synthesis of RNA upon DNA Templates, 35 Establishing the Genetic Code, 37 ESTABLISHING THE DIRECTION OF PROTEIN SYNTHESIS, 38 Start and Stop Signals Are Also Encoded within DNA, 40 THE ERA OF GENOMICS, 40 SUMMARY, 41 BIBLIOGRAPHY, 42 QUESTIONS, 42 xv xvi Detailed Contents PART 2: STRUCTURE AND STUDY OF MACROMOLECULES, 45 3 The Importance of Weak and Strong Chemical Bonds, 51 CHARACTERISTICS OF CHEMICAL BONDS, 51 Chemical Bonds Are Explainable in QuantumMechanical Terms, 52 Chemical-Bond Formation Involves a Change in the Form of Energy, 53 Equilibrium between Bond Making and Breaking, 53 THE CONCEPT OF FREE ENERGY, 54 Keq Is Exponentially Related to DG, 54 Covalent Bonds Are Very Strong, 54 WEAK BONDS IN BIOLOGICAL SYSTEMS, 55 Weak Bonds Have Energies between 1 and 7 kcal/mol, 55 Weak Bonds Are Constantly Made and Broken at Physiological Temperatures, 55 The Distinction between Polar and Nonpolar Molecules, 55 van der Waals Forces, 56 Hydrogen Bonds, 57 Some Ionic Bonds Are Hydrogen Bonds, 58 Weak Interactions Demand Complementary Molecular Surfaces, 58 Water Molecules Form Hydrogen Bonds, 59 Weak Bonds between Molecules in Aqueous Solutions, 59 Organic Molecules That Tend to Form Hydrogen Bonds Are Water Soluble, 60 Hydrophobic “Bonds” Stabilize Macromolecules, 60 ADVANCED CONCEPTS BOX 3-1 The Uniqueness of Molecular Shapes and the Concept of Selective Stickiness, 61 The Advantage of DG between 2 and 5 kcal/mol, 62 Weak Bonds Attach Enzymes to Substrates, 62 Weak Bonds Mediate Most Protein– DNA and Protein – Protein Interactions, 62 HIGH-ENERGY BONDS, 63 MOLECULES THAT DONATE ENERGY ARE THERMODYNAMICALLY UNSTABLE, 63 ENZYMES LOWER ACTIVATION ENERGIES IN BIOCHEMICAL REACTIONS, 65 FREE ENERGY IN BIOMOLECULES, 66 High-Energy Bonds Hydrolyze with Large Negative DG, 66 HIGH-ENERGY BONDS IN BIOSYNTHETIC REACTIONS, 67 Peptide Bonds Hydrolyze Spontaneously, 68 Coupling of Negative with Positive DG, 69 ACTIVATION OF PRECURSORS IN GROUP TRANSFER REACTIONS, 69 ATP Versatility in Group Transfer, 70 Activation of Amino Acids by Attachment of AMP, 70 Nucleic Acid Precursors Are Activated by the Presence of P P , 71 The Value of P P Release in Nucleic Acid Synthesis, 72 P P Splits Characterize Most Biosynthetic Reactions, 73 SUMMARY, 74 BIBLIOGRAPHY, 75 QUESTIONS, 75 4 The Structure of DNA, 77 DNA STRUCTURE, 78 DNA Is Composed of Polynucleotide Chains, 78 Each Base Has Its Preferred Tautomeric Form, 80 The Two Strands of the Double Helix Are Wound around Each Other in an Antiparallel Orientation, 81 The Two Chains of the Double Helix Have Complementary Sequences, 81 The Double Helix Is Stabilized by Base Pairing and Base Stacking, 82 Hydrogen Bonding Is Important for the Specificity of Base Pairing, 83 Bases Can Flip Out from the Double Helix, 83 DNA Is Usually a Right-Handed Double Helix, 83 KEY EXPERIMENTS BOX 4-1 DNA Has 10.5 bp per Turn of the Helix in Solution: The Mica Experiment, 84 Detailed Contents Topoisomerases Can Relax Supercoiled DNA, 97 Prokaryotes Have a Special Topoisomerase That Introduces Supercoils into DNA, 97 Topoisomerases Also Unknot and Disentangle DNA Molecules, 98 Topoisomerases Use a Covalent Protein –DNA Linkage to Cleave and Rejoin DNA Strands, 99 Topoisomerases Form an Enzyme Bridge and Pass DNA Segments through Each Other, 100 DNA Topoisomers Can Be Separated by Electrophoresis, 102 Ethidium Ions Cause DNA to Unwind, 102 KEY EXPERIMENTS BOX 4-3 Proving that DNA Has a Helical Periodicity of 10.5 bp per Turn from the Topological Properties of DNA Rings, 103 The Double Helix Has Minor and Major Grooves, 84 The Major Groove Is Rich in Chemical Information, 85 The Double Helix Exists in Multiple Conformations, 86 DNA Can Sometimes Form a Left-Handed Helix, 87 KEY EXPERIMENTS BOX 4-2 How Spots on an X-Ray Film Reveal the Structure of DNA, 88 DNA Strands Can Separate (Denature) and Reassociate, 89 Some DNA Molecules Are Circles, 92 DNA TOPOLOGY, 93 Linking Number Is an Invariant Topological Property of Covalently Closed, Circular DNA, 93 Linking Number Is Composed of Twist and Writhe, 93 Lk o Is the Linking Number of Fully Relaxed cccDNA under Physiological Conditions, 94 DNA in Cells Is Negatively Supercoiled, 95 Nucleosomes Introduce Negative Supercoiling in Eukaryotes, 96 xvii SUMMARY, 103 BIBLIOGRAPHY, 104 QUESTIONS, 104 5 The Structure and Versatility of RNA, 107 RNA CONTAINS RIBOSE AND URACIL AND IS USUALLY SINGLE-STRANDED, 107 DIRECTED EVOLUTION SELECTS RNAs THAT BIND SMALL MOLECULES, 114 RNA CHAINS FOLD BACK ON THEMSELVES TO FORM LOCAL REGIONS OF DOUBLE HELIX SIMILAR TO A-FORM DNA, 108 SOME RNAs ARE ENZYMES, 114 RNA CAN FOLD UP INTO COMPLEX TERTIARY STRUCTURES, 110 NUCLEOTIDE SUBSTITUTIONS IN COMBINATION WITH CHEMICAL PROBING PREDICT RNA STRUCTURE, 111 MEDICAL CONNECTIONS BOX 5-1 An RNA Switch Controls Protein Synthesis by Murine Leukemia Virus, 112 TECHNIQUES BOX 5-2 Creating an RNA Mimetic of the Green Fluorescent Protein by Directed Evolution, 115 The Hammerhead Ribozyme Cleaves RNA by the Formation of a 20 , 30 Cyclic Phosphate, 116 A Ribozyme at the Heart of the Ribosome Acts on a Carbon Center, 118 SUMMARY, 118 BIBLIOGRAPHY, 118 QUESTIONS, 118 6 The Structure of Proteins, 121 THE BASICS, 121 Amino Acids, 121 The Peptide Bond, 122 Polypeptide Chains, 123 Three Amino Acids with Special Conformational Properties, 124 ADVANCED CONCEPT BOX 6-1 Ramachandran Plot: Permitted Combinations of Main-Chain Torsion Angles f and c, 124 IMPORTANCE OF WATER, 125 PROTEIN STRUCTURE CAN BE DESCRIBED AT FOUR LEVELS, 126 PROTEIN DOMAINS, 130 Polypeptide Chains Typically Fold into One or More Domains, 130 ADVANCED CONCEPTS BOX 6-2 Glossary of Terms, 130 Basic Lessons from the Study of Protein Structures, 131 xviii Detailed Contents Classes of Protein Domains, 132 Linkers and Hinges, 133 Post-Translational Modifications, 133 ADVANCED CONCEPTS BOX 6-3 The Antibody Molecule as an Illustration of Protein Domains, 133 FROM AMINO-ACID SEQUENCE TO THREEDIMENSIONAL STRUCTURE, 134 Protein Folding, 134 PROTEINS AS AGENTS OF SPECIFIC MOLECULAR RECOGNITION, 137 Proteins That Recognize DNA Sequence, 137 Protein – Protein Interfaces, 140 Proteins That Recognize RNA, 141 ENZYMES: PROTEINS AS CATALYSTS, 141 REGULATION OF PROTEIN ACTIVITY, 142 KEY EXPERIMENTS BOX 6-4 Three-Dimensional SUMMARY, 143 Structure of a Protein Is Specified by Its Amino Acid Sequence (Anfinsen Experiment), 135 Predicting Protein Structure from Amino Acid Sequence, 135 BIBLIOGRAPHY, 144 QUESTIONS, 144 CONFORMATIONAL CHANGES IN PROTEINS, 136 7 Techniques of Molecular Biology, 147 NUCLEIC ACIDS: BASIC METHODS, 148 Gel Electrophoresis Separates DNA and RNA Molecules according to Size, 148 Restriction Endonucleases Cleave DNA Molecules at Particular Sites, 149 DNA Hybridization Can Be Used to Identify Specific DNA Molecules, 151 Hybridization Probes Can Identify Electrophoretically Separated DNAs and RNAs, 151 Isolation of Specific Segments of DNA, 153 DNA Cloning, 154 Vector DNA Can Be Introduced into Host Organisms by Transformation, 155 Libraries of DNA Molecules Can Be Created by Cloning, 156 Hybridization Can Be Used to Identify a Specific Clone in a DNA Library, 156 Chemical Synthesis of Defined DNA Sequences, 157 The Polymerase Chain Reaction Amplifies DNAs by Repeated Rounds of DNA Replication In Vitro, 158 Nested Sets of DNA Fragments Reveal Nucleotide Sequences, 158 TECHNIQUES BOX 7-1 Forensics and the Polymerase Chain Reaction, 160 Shotgun Sequencing a Bacterial Genome, 162 The Shotgun Strategy Permits a Partial Assembly of Large Genome Sequences, 162 KEY EXPERIMENTS BOX 7-2 Sequenators Are Used for High-Throughput Sequencing, 163 The Paired-End Strategy Permits the Assembly of Large-Genome Scaffolds, 165 The $1000 Human Genome Is within Reach, 167 GENOMICS, 168 Bioinformatics Tools Facilitate the Genome-Wide Identification of Protein-Coding Genes, 169 Whole-Genome Tiling Arrays Are Used to Visualize the Transcriptome, 169 Regulatory DNA Sequences Can Be Identified by Using Specialized Alignment Tools, 171 Genome Editing Is Used to Precisely Alter Complex Genomes, 172 PROTEINS, 173 Specific Proteins Can Be Purified from Cell Extracts, 173 Purification of a Protein Requires a Specific Assay, 173 Preparation of Cell Extracts Containing Active Proteins, 174 Proteins Can Be Separated from One Another Using Column Chromatography, 174 Separation of Proteins on Polyacrylamide Gels, 176 Antibodies Are Used to Visualize Electrophoretically Separated Proteins, 176 Protein Molecules Can Be Directly Sequenced, 177 PROTEOMICS, 179 Combining Liquid Chromatography with Mass Spectrometry Identifies Individual Proteins within a Complex Extract, 179 Proteome Comparisons Identify Important Differences between Cells, 181 Mass Spectrometry Can Also Monitor Protein Modification States, 181 Protein – Protein Interactions Can Yield Information regarding Protein Function, 182 Detailed Contents NUCLEIC ACID –PROTEIN INTERACTIONS, 182 The Electrophoretic Mobility of DNA Is Altered by Protein Binding, 183 DNA-Bound Protein Protects the DNA from Nucleases and Chemical Modification, 184 Chromatin Immunoprecipitation Can Detect Protein Association with DNA in the Cell, 185 xix Chromosome Conformation Capture Assays Are Used to Analyze Long-Range Interactions, 187 In Vitro Selection Can Be Used to Identify a Protein’s DNA- or RNA-Binding Site, 189 BIBLIOGRAPHY, 190 QUESTIONS, 190 PART 3: MAINTENANCE OF THE GENOME, 193 8 Genome Structure, Chromatin, and the Nucleosome, 199 GENOME SEQUENCE AND CHROMOSOME DIVERSITY, 200 Chromosomes Can Be Circular or Linear, 200 Every Cell Maintains a Characteristic Number of Chromosomes, 201 Genome Size Is Related to the Complexity of the Organism, 202 The E. coli Genome Is Composed Almost Entirely of Genes, 203 More Complex Organisms Have Decreased Gene Density, 204 Genes Make Up Only a Small Proportion of the Eukaryotic Chromosomal DNA, 205 The Majority of Human Intergenic Sequences Are Composed of Repetitive DNA, 207 CHROMOSOME DUPLICATION AND SEGREGATION, 208 Eukaryotic Chromosomes Require Centromeres, Telomeres, and Origins of Replication to Be Maintained during Cell Division, 208 Eukaryotic Chromosome Duplication and Segregation Occur in Separate Phases of the Cell Cycle, 210 Chromosome Structure Changes as Eukaryotic Cells Divide, 212 Sister-Chromatid Cohesion and Chromosome Condensation Are Mediated by SMC Proteins, 214 Mitosis Maintains the Parental Chromosome Number, 214 During Gap Phases, Cells Prepare for the Next Cell Cycle Stage and Check That the Previous Stage Is Completed Correctly, 217 Meiosis Reduces the Parental Chromosome Number, 217 Different Levels of Chromosome Structure Can Be Observed by Microscopy, 219 THE NUCLEOSOME, 220 Nucleosomes Are the Building Blocks of Chromosomes, 220 Histones Are Small, Positively Charged Proteins, 221 The Atomic Structure of the Nucleosome, 224 Histones Bind Characteristic Regions of DNA within the Nucleosome, 224 KEY EXPERIMENTS BOX 8-1 Micrococcal Nuclease and the DNA Associated with the Nucleosome, 226 Many DNA Sequence – Independent Contacts Mediate the Interaction between the Core Histones and DNA, 227 The Histone Amino-Terminal Tails Stabilize DNA Wrapping around the Octamer, 227 Wrapping of the DNA around the Histone Protein Core Stores Negative Superhelicity, 228 HIGHER-ORDER CHROMATIN STRUCTURE, 229 Heterochromatin and Euchromatin, 229 KEY EXPERIMENTS BOX 8-2 Nucleosomes and Superhelical Density, 230 Histone H1 Binds to the Linker DNA between Nucleosomes, 232 Nucleosome Arrays Can Form More Complex Structures: The 30-nm Fiber, 232 The Histone Amino-Terminal Tails Are Required for the Formation of the 30-nm Fiber, 234 Further Compaction of DNA Involves Large Loops of Nucleosomal DNA, 234 Histone Variants Alter Nucleosome Function, 234 REGULATION OF CHROMATIN STRUCTURE, 236 The Interaction of DNA with the Histone Octamer Is Dynamic, 236 Nucleosome-Remodeling Complexes Facilitate Nucleosome Movement, 237 Some Nucleosomes Are Found in Specific Positions: Nucleosome Positioning, 240 xx Detailed Contents The Amino-Terminal Tails of the Histones Are Frequently Modified, 241 Protein Domains in Nucleosome-Remodeling and -Modifying Complexes Recognize Modified Histones, 244 KEY EXPERIMENTS BOX 8-3 Determining Nucleosome Position in the Cell, 245 Specific Enzymes Are Responsible for Histone Modification, 248 Nucleosome Modification and Remodeling Work Together to Increase DNA Accessibility, 249 NUCLEOSOME ASSEMBLY, 249 Nucleosomes Are Assembled Immediately after DNA Replication, 249 Assembly of Nucleosomes Requires Histone “Chaperones”, 253 SUMMARY, 254 BIBLIOGRAPHY, 255 QUESTIONS, 255 9 The Replication of DNA, 257 THE CHEMISTRY OF DNA SYNTHESIS, 258 DNA Synthesis Requires Deoxynucleoside Triphosphates and a Primer:Template Junction, 258 DNA Is Synthesized by Extending the 30 End of the Primer, 259 Hydrolysis of Pyrophosphate Is the Driving Force for DNA Synthesis, 260 THE MECHANISM OF DNA POLYMERASE, 260 DNA Polymerases Use a Single Active Site to Catalyze DNA Synthesis, 260 TECHNIQUES BOX 9-1 Incorporation Assays Can Be Used to Measure Nucleic Acid and Protein Synthesis, 261 DNA Polymerases Resemble a Hand That Grips the Primer:Template Junction, 263 DNA Polymerases Are Processive Enzymes, 265 Exonucleases Proofread Newly Synthesized DNA, 267 MEDICAL CONNECTIONS BOX 9-2 Anticancer and Antiviral Agents Target DNA Replication, 268 THE REPLICATION FORK, 269 Both Strands of DNA Are Synthesized Together at the Replication Fork, 269 The Initiation of a New Strand of DNA Requires an RNA Primer, 270 RNA Primers Must Be Removed to Complete DNA Replication, 271 DNA Helicases Unwind the Double Helix in Advance of the Replication Fork, 272 DNA Helicase Pulls Single-Stranded DNA through a Central Protein Pore, 273 Single-Stranded DNA-Binding Proteins Stabilize ssDNA before Replication, 273 Topoisomerases Remove Supercoils Produced by DNA Unwinding at the Replication Fork, 275 Replication Fork Enzymes Extend the Range of DNA Polymerase Substrates, 275 THE SPECIALIZATION OF DNA POLYMERASES, 277 DNA Polymerases Are Specialized for Different Roles in the Cell, 277 Sliding Clamps Dramatically Increase DNA Polymerase Processivity, 278 Sliding Clamps Are Opened and Placed on DNA by Clamp Loaders, 281 ADVANCED CONCEPTS BOX 9-3 ATP Control of Protein Function: Loading a Sliding Clamp, 282 DNA SYNTHESIS AT THE REPLICATION FORK, 283 Interactions between Replication Fork Proteins Form the E. coli Replisome, 286 INITIATION OF DNA REPLICATION, 288 Specific Genomic DNA Sequences Direct the Initiation of DNA Replication, 288 The Replicon Model of Replication Initiation, 288 Replicator Sequences Include Initiator-Binding Sites and Easily Unwound DNA, 289 KEY EXPERIMENTS BOX 9-4 The Identification of Origins of Replication and Replicators, 290 BINDING AND UNWINDING: ORIGIN SELECTION AND ACTIVATION BY THE INITIATOR PROTEIN, 293 Protein – Protein and Protein – DNA Interactions Direct the Initiation Process, 293 ADVANCED CONCEPTS BOX 9-5 E. coli DNA Replication Is Regulated by DnaA.ATP Levels and SeqA, 294 Eukaryotic Chromosomes Are Replicated Exactly Once per Cell Cycle, 297 Helicase Loading Is the First Step in the Initiation of Replication in Eukaryotes, 298 Helicase Loading and Activation Are Regulated to Allow Only a Single Round of Replication during Each Cell Cycle, 300 Detailed Contents MEDICAL CONNECTIONS BOX 9-6 Aging, Cancer, and Similarities between Eukaryotic and Prokaryotic DNA Replication Initiation, 301 the Telomere Hypothesis, 307 Telomere-Binding Proteins Regulate Telomerase Activity and Telomere Length, 307 Telomere-Binding Proteins Protect Chromosome Ends, 308 FINISHING REPLICATION, 302 Type II Topoisomerases Are Required to Separate Daughter DNA Molecules, 303 Lagging-Strand Synthesis Is Unable to Copy the Extreme Ends of Linear Chromosomes, 303 Telomerase Is a Novel DNA Polymerase That Does Not Require an Exogenous Template, 305 Telomerase Solves the End Replication Problem by Extending the 30 End of the Chromosome, 305 xxi SUMMARY, 310 BIBLIOGRAPHY, 311 QUESTIONS, 312 10 The Mutability and Repair of DNA, 313 REPLICATION ERRORS AND THEIR REPAIR, 314 The Nature of Mutations, 314 Some Replication Errors Escape Proofreading, 315 MEDICAL CONNECTIONS BOX 10-1 Expansion of Triple Repeats Causes Disease, 316 Mismatch Repair Removes Errors That Escape Proofreading, 316 DNA DAMAGE, 320 DNA Undergoes Damage Spontaneously from Hydrolysis and Deamination, 320 MEDICAL CONNECTIONS BOX 10-2 The Ames Test, 321 DNA Is Damaged by Alkylation, Oxidation, and Radiation, 322 ADVANCED CONCEPTS BOX 10-3 Quantitation of DNA Damage and Its Effects on Cellular Survival and Mutagenesis, 323 Mutations Are Also Caused by Base Analogs and Intercalating Agents, 323 REPAIR AND TOLERANCE OF DNA DAMAGE, 324 Direct Reversal of DNA Damage, 325 Base Excision Repair Enzymes Remove Damaged Bases by a Base-Flipping Mechanism, 326 Nucleotide Excision Repair Enzymes Cleave Damaged DNA on Either Side of the Lesion, 328 MEDICAL CONNECTIONS BOX 10-4 Linking Nucleotide Excision Repair and Translesion Synthesis to a Genetic Disorder in Humans, 330 Recombination Repairs DNA Breaks by Retrieving Sequence Information from Undamaged DNA, 330 DSBs in DNA Are Also Repaired by Direct Joining of Broken Ends, 331 MEDICAL CONNECTIONS BOX 10-5 Nonhomologous End Joining, 332 Translesion DNA Synthesis Enables Replication to Proceed across DNA Damage, 333 ADVANCED CONCEPTS BOX 10-6 The Y Family of DNA Polymerases, 336 SUMMARY, 338 BIBLIOGRAPHY, 338 QUESTIONS, 339 11 Homologous Recombination at the Molecular Level, 341 DNA BREAKS ARE COMMON AND INITIATE RECOMBINATION, 342 MODELS FOR HOMOLOGOUS RECOMBINATION, 342 Strand Invasion Is a Key Early Step in Homologous Recombination, 344 Resolving Holliday Junctions Is a Key Step to Finishing Genetic Exchange, 346 The Double-Strand Break –Repair Model Describes Many Recombination Events, 346 HOMOLOGOUS RECOMBINATION PROTEIN MACHINES, 349 ADVANCED CONCEPTS BOX 11-1 How to Resolve a Recombination Intermediate with Two Holliday Junctions, 350 The RecBCD Helicase/Nuclease Processes Broken DNA Molecules for Recombination, 351 Chi Sites Control RecBCD, 354 RecA Protein Assembles on Single-Stranded DNA and Promotes Strand Invasion, 355 xxii Detailed Contents Newly Base-Paired Partners Are Established within the RecA Filament, 356 RecA Homologs Are Present in All Organisms, 359 The RuvAB Complex Specifically Recognizes Holliday Junctions and Promotes Branch Migration, 359 RuvC Cleaves Specific DNA Strands at the Holliday Junction to Finish Recombination, 361 HOMOLOGOUS RECOMBINATION IN EUKARYOTES, 362 Homologous Recombination Has Additional Functions in Eukaryotes, 362 Homologous Recombination Is Required for Chromosome Segregation during Meiosis, 362 Programmed Generation of Double-Stranded DNA Breaks Occurs during Meiosis, 363 MRX Protein Processes the Cleaved DNA Ends for Assembly of the RecA-Like Strand-Exchange Proteins, 364 Dmc1 Is a RecA-Like Protein That Specifically Functions in Meiotic Recombination, 366 Many Proteins Function Together to Promote Meiotic Recombination, 366 MEDICAL CONNECTIONS BOX 11-2 The Product of the Tumor Suppressor Gene BRCA2 Interacts with Rad51 Protein and Controls Genome Stability, 367 MEDICAL CONNECTIONS BOX 11-3 Proteins Associated with Premature Aging and Cancer Promote an Alternative Pathway for Holliday Junction Processing, 368 MATING-TYPE SWITCHING, 369 Mating-Type Switching Is Initiated by a Site-Specific Double-Strand Break, 370 Mating-Type Switching Is a Gene Conversion Event and Not Associated with Crossing Over, 370 GENETIC CONSEQUENCES OF THE MECHANISM OF HOMOLOGOUS RECOMBINATION, 371 One Cause of Gene Conversion Is DNA Repair during Recombination, 373 SUMMARY, 374 BIBLIOGRAPHY, 375 QUESTIONS, 376 12 Site-Specific Recombination and Transposition of DNA, 377 CONSERVATIVE SITE-SPECIFIC RECOMBINATION, 378 Site-Specific Recombination Occurs at Specific DNA Sequences in the Target DNA, 378 Site-Specific Recombinases Cleave and Rejoin DNA Using a Covalent Protein – DNA Intermediate, 380 Serine Recombinases Introduce Double-Strand Breaks in DNA and Then Swap Strands to Promote Recombination, 382 Structure of the Serine Recombinase – DNA Complex Indicates that Subunits Rotate to Achieve Strand Exchange, 383 Tyrosine Recombinases Break and Rejoin One Pair of DNA Strands at a Time, 383 Structures of Tyrosine Recombinases Bound to DNA Reveal the Mechanism of DNA Exchange, 384 MEDICAL CONNECTIONS BOX 12-1 Application of Site-Specific Recombination to Genetic Engineering, 386 BIOLOGICAL ROLES OF SITE-SPECIFIC RECOMBINATION, 386 l Integrase Promotes the Integration and Excision of a Viral Genome into the Host-Cell Chromosome, 386 Bacteriophage l Excision Requires a New DNA-Bending Protein, 389 The Hin Recombinase Inverts a Segment of DNA Allowing Expression of Alternative Genes, 389 Hin Recombination Requires a DNA Enhancer, 390 Recombinases Convert Multimeric Circular DNA Molecules into Monomers, 391 There Are Other Mechanisms to Direct Recombination to Specific Segments of DNA, 391 ADVANCED CONCEPTS BOX 12-2 The Xer Recombinase Catalyzes the Monomerization of Bacterial Chromosomes and of Many Bacterial Plasmids, 392 TRANSPOSITION, 393 Some Genetic Elements Move to New Chromosomal Locations by Transposition, 393 There Are Three Principal Classes of Transposable Elements, 395 DNA Transposons Carry a Transposase Gene, Flanked by Recombination Sites, 395 Transposons Exist as Both Autonomous and Nonautonomous Elements, 396 Virus-Like Retrotransposons and Retroviruses Carry Terminal Repeat Sequences and Two Genes Important for Recombination, 396 Poly-A Retrotransposons Look Like Genes, 396 DNA Transposition by a Cut-and-Paste Mechanism, 397 Detailed Contents The Intermediate in Cut-and-Paste Transposition is Finished by Gap Repair, 398 There Are Multiple Mechanisms for Cleaving the Nontransferred Strand during DNA Transposition, 399 DNA Transposition by a Replicative Mechanism, 401 Virus-Like Retrotransposons and Retroviruses Move Using an RNA Intermediate, 403 DNA Transposases and Retroviral Integrases Are Members of a Protein Superfamily, 403 Poly-A Retrotransposons Move by a “Reverse Splicing” Mechanism, 405 EXAMPLES OF TRANSPOSABLE ELEMENTS AND THEIR REGULATION, 406 KEY EXPERIMENTS BOX 12-3 Maize Elements and Discovery of Transposons, 408 IS4 Family Transposons Are Compact Elements with Multiple Mechanisms for Copy Number Control, 409 Phage Mu Is an Extremely Robust Transposon, 411 Mu Uses Target Immunity to Avoid Transposing into Its Own DNA, 411 Tc1/mariner Elements Are Highly Successful DNA Elements in Eukaryotes, 411 ADVANCED CONCEPTS BOX 12-4 Mechanism of Transposition Target Immunity, 413 Yeast Ty Elements Transpose into Safe Havens in the Genome, 414 LINEs Promote Their Own Transposition and Even Transpose Cellular RNAs, 414 V(D)J RECOMBINATION, 416 The Early Events in V(D)J Recombination Occur by a Mechanism Similar to Transposon Excision, 418 SUMMARY, 420 BIBLIOGRAPHY, 420 QUESTIONS, 421 PART 4: EXPRESSION OF THE GENOME, 423 13 Mechanisms of Transcription, 429 RNA POLYMERASES AND THE TRANSCRIPTION CYCLE, 430 RNA Polymerases Come in Different Forms but Share Many Features, 430 Transcription by RNA Polymerase Proceeds in a Series of Steps, 432 Transcription Initiation Involves Three Defined Steps, 434 THE TRANSCRIPTION CYCLE IN BACTERIA, 434 Bacterial Promoters Vary in Strength and Sequence but Have Certain Defining Features, 434 TECHNIQUES BOX 13-1 Consensus Sequences, 436 The s Factor Mediates Binding of Polymerase to the Promoter, 437 Transition to the Open Complex Involves Structural Changes in RNA Polymerase and in the Promoter DNA, 438 Transcription Is Initiated by RNA Polymerase without the Need for a Primer, 440 During Initial Transcription, RNA Polymerase Remains Stationary and Pulls Downstream DNA into Itself, 441 Promoter Escape Involves Breaking Polymerase – Promoter Interactions and Polymerase Core– s Interactions, 442 xxiii The Elongating Polymerase Is a Processive Machine That Synthesizes and Proofreads RNA, 442 ADVANCED CONCEPTS BOX 13-2 The Single-Subunit RNA Polymerases, 443 RNA Polymerase Can Become Arrested and Need Removing, 445 Transcription Is Terminated by Signals within the RNA Sequence, 445 TRANSCRIPTION IN EUKARYOTES, 448 RNA Polymerase II Core Promoters Are Made Up of Combinations of Different Classes of Sequence Element, 448 RNA Polymerase II Forms a Preinitiation Complex with General Transcription Factors at the Promoter, 449 Promoter Escape Requires Phosphorylation of the Polymerase “Tail,” 449 TBP Binds to and Distorts DNA Using a b Sheet Inserted into the Minor Groove, 451 The Other General Transcription Factors Also Have Specific Roles in Initiation, 452 In Vivo, Transcription Initiation Requires Additional Proteins, Including the Mediator Complex, 453 xxiv Detailed Contents Mediator Consists of Many Subunits, Some Conserved from Yeast to Human, 454 A New Set of Factors Stimulates Pol II Elongation and RNA Proofreading, 455 Elongating RNA Polymerase Must Deal with Histones in Its Path, 456 Elongating Polymerase Is Associated with a New Set of Protein Factors Required for Various Types of RNA Processing, 457 Transcription Termination Is Linked to RNA Destruction by a Highly Processive RNase, 460 5' 3' A A 5' 3' TRANSCRIPTION BY RNA POLYMERASES I AND III, 462 RNA Pol I and Pol III Recognize Distinct Promoters but Still Require TBP, 462 Pol I Transcribes Just the rRNA Genes, 462 Pol III Promoters Are Found Downstream from the Transcription Start Site, 463 SUMMARY, 463 BIBLIOGRAPHY, 464 QUESTIONS, 465 14 RNA Splicing, 467 3' THE CHEMISTRY OF RNA SPLICING, 469 Sequences within the RNA Determine Where Splicing Occurs, 469 The Intron Is Removed in a Form Called a Lariat as the Flanking Exons Are Joined, 470 KEY EXPERIMENTS BOX 14-1 Adenovirus and the Discovery of Splicing, 471 THE SPLICEOSOME MACHINERY, 473 RNA Splicing Is Performed by a Large Complex Called the Spliceosome, 473 SPLICING PATHWAYS, 474 Assembly, Rearrangements, and Catalysis within the Spliceosome: The Splicing Pathway, 474 Spliceosome Assembly Is Dynamic and Variable and Its Disassembly Ensures That the Splicing Reaction Goes Only Forward in the Cell, 476 Self-Splicing Introns Reveal That RNA Can Catalyze RNA Splicing, 477 Group I Introns Release a Linear Intron Rather Than a Lariat, 478 KEY EXPERIMENTS BOX 14-2 Converting Group I Introns into Ribozymes, 479 How Does the Spliceosome Find the Splice Sites Reliably?, 480 VARIANTS OF SPLICING, 482 Exons from Different RNA Molecules Can Be Fused by Trans-Splicing, 482 A Small Group of Introns Is Spliced by an Alternative Spliceosome Composed of a Different Set of snRNPs, 483 ALTERNATIVE SPLICING, 483 Single Genes Can Produce Multiple Products by Alternative Splicing, 483 Several Mechanisms Exist to Ensure Mutually Exclusive Splicing, 486 The Curious Case of the Drosophila Dscam Gene: Mutually Exclusive Splicing on a Grand Scale, 487 Mutually Exclusive Splicing of Dscam Exon 6 Cannot Be Accounted for by Any Standard Mechanism and Instead Uses a Novel Strategy, 488 KEY EXPERIMENTS BOX 14-3 Identification of Docking Site and Selector Sequences, 490 Alternative Splicing Is Regulated by Activators and Repressors, 491 Regulation of Alternative Splicing Determines the Sex of Flies, 493 An Alternative Splicing Switch Lies at the Heart of Pluripotency, 495 EXON SHUFFLING, 497 Exons Are Shuffled by Recombination to Produce Genes Encoding New Proteins, 497 MEDICAL CONNECTIONS BOX 14-4 Defects in Pre-mRNA Splicing Cause Human Disease, 497 RNA EDITING, 500 RNA Editing Is Another Way of Altering the Sequence of an mRNA, 500 Guide RNAs Direct the Insertion and Deletion of Uridines, 501 MEDICAL CONNECTIONS BOX 14-5 Deaminases and HIV, 503 mRNA TRANSPORT, 503 Once Processed, mRNA Is Packaged and Exported from the Nucleus into the Cytoplasm for Translation, 503 SUMMARY, 505 BIBLIOGRAPHY, 506 QUESTIONS, 507 Detailed Contents xxv 15 Translation, 509 MESSENGER RNA, 510 Polypeptide Chains Are Specified by Open Reading Frames, 510 Prokaryotic mRNAs Have a Ribosome-Binding Site That Recruits the Translational Machinery, 512 Eukaryotic mRNAs Are Modified at their 50 and 30 Ends to Facilitate Translation, 512 TRANSFER RNA, 513 tRNAs Are Adaptors between Codons and Amino Acids, 513 ADVANCED CONCEPTS BOX 15-1 CCA-Adding Enzymes: Synthesizing RNA without a Template, 513 tRNAs Share a Common Secondary Structure That Resembles a Cloverleaf, 514 tRNAs Have an L-Shaped Three-Dimensional Structure, 514 ATTACHMENT OF AMINO ACIDS TO tRNA, 515 tRNAs Are Charged by the Attachment of an Amino Acid to the 30 -Terminal Adenosine Nucleotide via a High-Energy Acyl Linkage, 515 Aminoacyl-tRNA Synthetases Charge tRNAs in Two Steps, 515 Each Aminoacyl-tRNA Synthetase Attaches a Single Amino Acid to One or More tRNAs, 515 tRNA Synthetases Recognize Unique Structural Features of Cognate tRNAs, 517 Aminoacyl-tRNA Formation Is Very Accurate, 518 Some Aminoacyl-tRNA Synthetases Use an Editing Pocket to Charge tRNAs with High Accuracy, 518 The Ribosome Is Unable to Discriminate between Correctly and Incorrectly Charged tRNAs, 519 THE RIBOSOME, 519 ADVANCED CONCEPTS BOX 15-2 Selenocysteine, 520 The Ribosome Is Composed of a Large and a Small Subunit, 521 The Large and Small Subunits Undergo Association and Dissociation during Each Cycle of Translation, 522 New Amino Acids Are Attached to the Carboxyl Terminus of the Growing Polypeptide Chain, 523 Peptide Bonds Are Formed by Transfer of the Growing Polypeptide Chain from One tRNA to Another, 524 Ribosomal RNAs Are Both Structural and Catalytic Determinants of the Ribosome, 524 The Ribosome Has Three Binding Sites for tRNA, 525 Channels through the Ribosome Allow the mRNA and Growing Polypeptide to Enter and/or Exit the Ribosome, 527 INITIATION OF TRANSLATION, 528 Prokaryotic mRNAs Are Initially Recruited to the Small Subunit by Base Pairing to rRNA, 528 A Specialized tRNA Charged with a Modified Methionine Binds Directly to the Prokaryotic Small Subunit, 528 Three Initiation Factors Direct the Assembly of an Initiation Complex That Contains mRNA and the Initiator tRNA, 529 Eukaryotic Ribosomes Are Recruited to the mRNA by the 50 Cap, 530 Translation Initiation Factors Hold Eukaryotic mRNAs in Circles, 532 ADVANCED CONCEPTS BOX 15-3 uORFs and IRESs: Exceptions That Prove the Rule, 533 The Start Codon Is Found by Scanning Downstream from the 50 End of the mRNA, 535 TRANSLATION ELONGATION, 535 Aminoacyl-tRNAs Are Delivered to the A-Site by Elongation Factor EF-Tu, 537 The Ribosome Uses Multiple Mechanisms to Select against Incorrect Aminoacyl-tRNAs, 537 The Ribosome Is a Ribozyme, 538 Peptide-Bond Formation Initiates Translocation in the Large Subunit, 541 EF-G Drives Translocation by Stabilizing Intermediates in Translocation, 542 EF-Tu– GDP and EF-G– GDP Must Exchange GDP for GTP before Participating in a New Round of Elongation, 543 A Cycle of Peptide-Bond Formation Consumes Two Molecules of GTP and One Molecule of ATP, 543 TERMINATION OF TRANSLATION, 544 Release Factors Terminate Translation in Response to Stop Codons, 544 Short Regions of Class I Release Factors Recognize Stop Codons and Trigger Release of the Peptidyl Chain, 544 ADVANCED CONCEPTS BOX 15-4 GTP-Binding Proteins, Conformational Switching, and the Fidelity and Ordering of the Events of Translation, 546 GDP/GTP Exchange and GTP Hydrolysis Control the Function of the Class II Release Factor, 547 The Ribosome Recycling Factor Mimics a tRNA, 548 REGULATION OF TRANSLATION, 549 Protein or RNA Binding near the Ribosome-Binding Site Negatively Regulates Bacterial Translation Initiation, 549 Regulation of Prokaryotic Translation: Ribosomal Proteins Are Translational Repressors of Their Own Synthesis, 551 xxvi Detailed Contents MEDICAL CONNECTIONS BOX 15-5 Antibiotics Arrest Cell Division by Blocking Specific Steps in Translation, 552 Global Regulators of Eukaryotic Translation Target Key Factors Required for mRNA Recognition and Initiator tRNA Ribosome Binding, 556 Spatial Control of Translation by mRNA-Specific 4E-BPs, 556 An Iron-Regulated, RNA-Binding Protein Controls Translation of Ferritin, 557 Translation of the Yeast Transcriptional Activator Gcn4 Is Controlled by Short Upstream ORFs and Ternary Complex Abundance, 558 TECHNIQUES BOX 15-6 Ribosome and Polysome Profiling, 561 TRANSLATION-DEPENDENT REGULATION OF mRNA AND PROTEIN STABILITY, 563 The SsrA RNA Rescues Ribosomes That Translate Broken mRNAs, 563 MEDICAL CONNECTIONS BOX 15-7 A Frontline Drug in Tuberculosis Therapy Targets SsrA Tagging, 565 Eukaryotic Cells Degrade mRNAs That Are Incomplete or Have Premature Stop Codons, 565 SUMMARY, 567 BIBLIOGRAPHY, 570 QUESTIONS, 570 16 The Genetic Code, 573 THE CODE IS DEGENERATE, 573 Perceiving Order in the Makeup of the Code, 575 Wobble in the Anticodon, 575 Three Codons Direct Chain Termination, 577 How the Code Was Cracked, 577 Stimulation of Amino Acid Incorporation by Synthetic mRNAs, 578 Poly-U Codes for Polyphenylalanine, 579 Mixed Copolymers Allowed Additional Codon Assignments, 579 Transfer RNA Binding to Defined Trinucleotide Codons, 579 Codon Assignments from Repeating Copolymers, 581 THREE RULES GOVERN THE GENETIC CODE, 582 Three Kinds of Point Mutations Alter the Genetic Code, 582 Genetic Proof That the Gode Is Read in Units of Three, 583 SUPPRESSOR MUTATIONS CAN RESIDE IN THE SAME OR A DIFFERENT GENE, 584 Intergenic Suppression Involves Mutant tRNAs, 584 Nonsense Suppressors Also Read Normal Termination Signals, 585 Proving the Validity of the Genetic Code, 586 THE CODE IS NEARLY UNIVERSAL, 587 ADVANCED CONCEPTS BOX 16-1 Expanding the Genetic Code, 589 SUMMARY, 590 BIBLIOGRAPHY, 590 QUESTIONS, 591 17 The Origin and Early Evolution of Life, 593 WHEN DID LIFE ARISE ON EARTH?, 594 WHAT WAS THE BASIS FOR PREBIOTIC ORGANIC CHEMISTRY?, 595 DOES DARWINIAN EVOLUTION REQUIRE SELF-REPLICATING PROTOCELLS?, 603 DID LIFE ARISE ON EARTH?, 606 DID LIFE EVOLVE FROM AN RNA WORLD?, 599 SUMMARY, 607 CAN SELF-REPLICATING RIBOZYMES BE CREATED BY DIRECTED EVOLUTION?, 599 BIBLIOGRAPHY, 607 QUESTIONS, 607 Detailed Contents xxvii PART 5: REGULATION, 609 18 Transcriptional Regulation in Prokaryotes, 615 PRINCIPLES OF TRANSCRIPTIONAL REGULATION, 615 Gene Expression Is Controlled by Regulatory Proteins, 615 Most Activators and Repressors Act at the Level of Transcription Initiation, 616 Many Promoters Are Regulated by Activators That Help RNA Polymerase Bind DNA and by Repressors That Block That Binding, 616 Some Activators and Repressors Work by Allostery and Regulate Steps in Transcriptional Initiation after RNA Polymerase Binding, 618 Action at a Distance and DNA Looping, 618 Cooperative Binding and Allostery Have Many Roles in Gene Regulation, 619 Antitermination and Beyond: Not All of Gene Regulation Targets Transcription Initiation, 620 REGULATION OF TRANSCRIPTION INITIATION: EXAMPLES FROM PROKARYOTES, 620 An Activator and a Repressor Together Control the lac Genes, 620 CAP and Lac Repressor Have Opposing Effects on RNA Polymerase Binding to the lac Promoter, 622 CAP Has Separate Activating and DNA-Binding Surfaces, 622 CAP and Lac Repressor Bind DNA Using a Common Structural Motif, 623 KEY EXPERIMENTS BOX 18-1 Activator Bypass Experiments, 624 The Activities of Lac Repressor and CAP Are Controlled Allosterically by Their Signals, 626 Combinatorial Control: CAP Controls Other Genes As Well, 627 KEY EXPERIMENTS BOX 18-2 Jacob, Monod, and the Ideas behind Gene Regulation, 628 Alternative s Factors Direct RNA Polymerase to Alternative Sets of Promoters, 630 NtrC and MerR: Transcriptional Activators That Work by Allostery Rather than by Recruitment, 630 NtrC Has ATPase Activity and Works from DNA Sites Far from the Gene, 631 MerR Activates Transcription by Twisting Promoter DNA, 632 Some Repressors Hold RNA Polymerase at the Promoter Rather than Excluding It, 633 AraC and Control of the araBAD Operon by Antiactivation, 634 MEDICAL CONNECTIONS 18-3 Blocking Virulence by Silencing Pathways of Intercellular Communication, 635 THE CASE OF BACTERIOPHAGE l: LAYERS OF REGULATION, 636 Alternative Patterns of Gene Expression Control Lytic and Lysogenic Growth, 636 Regulatory Proteins and Their Binding Sites, 638 l Repressor Binds to Operator Sites Cooperatively, 639 Repressor and Cro Bind in Different Patterns to Control Lytic and Lysogenic Growth, 640 ADVANCED CONCEPTS BOX 18-4 Concentration, Affinity, and Cooperative Binding, 641 Lysogenic Induction Requires Proteolytic Cleavage of l Repressor, 642 Negative Autoregulation of Repressor Requires Long-Distance Interactions and a Large DNA Loop, 643 Another Activator, l CII, Controls the Decision between Lytic and Lysogenic Growth upon Infection of a New Host, 644 KEY EXPERIMENTS BOX 18-5 Evolution of the l Switch, 645 The Number of Phage Particles Infecting a Given Cell Affects Whether the Infection Proceeds Lytically or Lysogenically, 647 Growth Conditions of E. coli Control the Stability of CII Protein and Thus the Lytic/Lysogenic Choice, 648 Transcriptional Antitermination in l Development, 648 KEY EXPERIMENTS BOX 18-6 Genetic Approaches That Identified Genes Involved in the Lytic/Lysogenic Choice, 649 Retroregulation: An Interplay of Controls on RNA Synthesis and Stability Determines int Gene Expression, 651 SUMMARY, 652 BIBLIOGRAPHY, 653 QUESTIONS, 654 xxviii Detailed Contents 19 Transcriptional Regulation in Eukaryotes, 657 CONSERVED MECHANISMS OF TRANSCRIPTIONAL REGULATION FROM YEAST TO MAMMALS, 659 Activators Have Separate DNA-Binding and Activating Functions, 660 Eukaryotic Regulators Use a Range of DNA-Binding Domains, But DNA Recognition Involves the Same Principles as Found in Bacteria, 661 Activating Regions Are Not Well-Defined Structures, 663 TECHNIQUES BOX 19-1 The Two-Hybrid Assay, 664 RECRUITMENT OF PROTEIN COMPLEXES TO GENES BY EUKARYOTIC ACTIVATORS, 665 Activators Recruit the Transcriptional Machinery to the Gene, 665 TECHNIQUES BOX 19-2 The ChIP-Chip and ChIP-Seq Assays Are the Best Method for Identifying Enhancers, 666 Activators Also Recruit Nucleosome Modifiers That Help the Transcriptional Machinary Bind at the Promoter or Initiate Transcription, 667 Activators Recruit Additional Factors Needed for Efficient Initiation or Elongation at Some Promoters, 669 MEDICAL CONNECTIONS BOX 19-3 Histone Modifications, Transcription Elongation, and Leukemia, 670 Action at a Distance: Loops and Insulators, 672 Appropriate Regulation of Some Groups of Genes Requires Locus Control Regions, 673 SIGNAL INTEGRATION AND COMBINATORIAL CONTROL, 675 Activators Work Synergistically to Integrate Signals, 675 Signal Integration: The HO Gene Is Controlled by Two Regulators—One Recruits Nucleosome Modifiers, and the Other Recruits Mediator, 675 Signal Integration: Cooperative Binding of Activators at the Human b-Interferon Gene, 676 Combinatorial Control Lies at the Heart of the Complexity and Diversity of Eukaryotes, 678 Combinatorial Control of the Mating-Type Genes from S. cerevisiae, 680 TRANSCRIPTIONAL REPRESSORS, 681 SIGNAL TRANSDUCTION AND THE CONTROL OF TRANSCRIPTIONAL REGULATORS, 682 Signals Are Often Communicated to Transcriptional Regulators through Signal Transduction Pathways, 682 KEY EXPERIMENTS BOX 19-4 Evolution of a Regulatory Circuit, 683 Signals Control the Activities of Eukaryotic Transcriptional Regulators in a Variety of Ways, 686 GENE “SILENCING” BY MODIFICATION OF HISTONES AND DNA, 687 Silencing in Yeast Is Mediated by Deacetylation and Methylation of Histones, 688 In Drosophila, HP1 Recognizes Methylated Histones and Condenses Chromatin, 689 Repression by Polycomb Also Uses Histone Methylation, 690 ADVANCED CONCEPTS BOX 19-5 Is There a Histone Code?, 691 DNA Methylation Is Associated with Silenced Genes in Mammalian Cells, 692 EPIGENETIC GENE REGULATION, 694 Some States of Gene Expression Are Inherited through Cell Division Even When the Initiating Signal Is No Longer Present, 694 MEDICAL CONNECTIONS BOX 19-6 Transcriptional Repression and Human Disease, 696 SUMMARY, 697 BIBLIOGRAPHY, 698 QUESTIONS, 699 20 Regulatory RNAs, 701 REGULATION BY RNAS IN BACTERIA, 701 Riboswitches Reside within the Transcripts of Genes Whose Expression They Control through Changes in Secondary Structure, 703 RNAs as Defense Agents in Prokaryotes and Archaea, 705 CRISPRs Are a Record of Infections Survived and Resistance Gained, 706 ADVANCED CONCEPTS BOX 20-1 Amino Acid Biosynthetic Operons Are Controlled by Attenuation, 707 Spacer Sequences Are Acquired from Infecting Viruses, 710 Detailed Contents A CRISPR Is Transcribed as a Single Long RNA, Which Is Then Processed into Shorter RNA Species That Target Destruction of Invading DNA or RNA, 710 REGULATORY RNAs ARE WIDESPREAD IN EUKARYOTES, 711 Short RNAs That Silence Genes Are Produced from a Variety of Sources and Direct the Silencing of Genes in Three Different Ways, 712 SYNTHESIS AND FUNCTION OF miRNA MOLECULES, 714 miRNAs Have a Characteristic Structure That Assists in Identifying Them and Their Target Genes, 714 An Active miRNA Is Generated through a Two-Step Nucleolytic Processing, 716 Dicer Is the Second RNA-Cleaving Enzyme Involved in miRNA Production and the Only One Needed for siRNA Production, 717 SILENCING GENE EXPRESSION BY SMALL RNAs, 718 Incorporation of a Guide Strand RNA into RISC Makes the Mature Complex That Is Ready to Silence Gene Expression, 718 xxix Small RNAs Can Transcriptionally Silence Genes by Directing Chromatin Modification, 719 RNAi Is a Defense Mechanism That Protects against Viruses and Transposons, 721 KEY EXPERIMENTS BOX 20-2 Discovery of miRNAs and RNAi, 722 RNAi Has Become a Powerful Tool for Manipulating Gene Expression, 725 MEDICAL CONNECTIONS BOX 20-3 microRNAs and Human Disease, 727 LONG NON-CODING RNAS AND X-INACTIVATION, 728 Long Non-Coding RNAs Have Many Roles in Gene Regulation, Including Cis and Trans Effects on Transcription, 728 X-Inactivation Creates Mosaic Individuals, 728 Xist Is a Long Non-Coding RNA That Inactivates a Single X Chromosome in Female Mammals, 729 SUMMARY, 730 BIBLIOGRAPHY, 731 QUESTIONS, 732 21 Gene Regulation in Development and Evolution, 733 MEDICAL CONNECTIONS BOX 21-1 Formation of iPS Cells, 734 THREE STRATEGIES BY WHICH CELLS ARE INSTRUCTED TO EXPRESS SPECIFIC SETS OF GENES DURING DEVELOPMENT, 735 Some mRNAs Become Localized within Eggs and Embryos Because of an Intrinsic Polarity in the Cytoskeleton, 735 Cell-to-Cell Contact and Secreted Cell-Signaling Molecules Both Elicit Changes in Gene Expression in Neighboring Cells, 736 Gradients of Secreted Signaling Molecules Can Instruct Cells to Follow Different Pathways of Development Based on Their Location, 737 EXAMPLES OF THE THREE STRATEGIES FOR ESTABLISHING DIFFERENTIAL GENE EXPRESSION, 738 The Localized Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO Gene, 738 A Localized mRNA Initiates Muscle Differentiation in the Sea Squirt Embryo, 740 ADVANCED CONCEPTS BOX 21-2 Review of Cytoskeleton: Asymmetry and Growth, 741 Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium, Bacillus subtilis, 743 A Skin – Nerve Regulatory Switch Is Controlled by Notch Signaling in the Insect Central Nervous System, 743 A Gradient of the Sonic Hedgehog Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural Tube, 744 THE MOLECULAR BIOLOGY OF DROSOPHILA EMBRYOGENESIS, 746 An Overview of Drosophila Embryogenesis, 746 A Regulatory Gradient Controls Dorsoventral Patterning of the Drosophila Embryo, 747 ADVANCED CONCEPTS BOX 21-3 Overview of Drosophila Development, 748 Segmentation Is Initiated by Localized RNAs at the Anterior and Posterior Poles of the Unfertilized Egg, 751 KEY EXPERIMENTS BOX 21-4 Activator Synergy, 752 Bicoid and Nanos Regulate hunchback, 753 Multiple Enhancers Ensure Precision of hunchback Regulation, 754 The Gradient of Hunchback Repressor Establishes Different Limits of Gap Gene Expression, 754 MEDICAL CONNECTIONS BOX 21-5 Stem Cell Niche, 755 ADVANCED CONCEPTS BOX 21-6 Gradient Thresholds, 757 xxx Detailed Contents Hunchback and Gap Proteins Produce Segmentation Stripes of Gene Expression, 758 KEY EXPERIMENTS BOX 21-7 cis-Regulatory Sequences in Animal Development and Evolution, 759 Gap Repressor Gradients Produce Many Stripes of Gene Expression, 760 Short-Range Transcriptional Repressors Permit Different Enhancers to Work Independently of One Another within the Complex eve Regulatory Region, 761 HOMEOTIC GENES: AN IMPORTANT CLASS OF DEVELOPMENTAL REGULATORS, 762 Changes in Homeotic Gene Expression Are Responsible for Arthropod Diversity, 763 Changes in Ubx Expression Explain Modifications in Limbs among the Crustaceans, 763 ADVANCED CONCEPTS BOX 21-8 Homeotic Genes of Drosophila Are Organized in Special Chromosome Clusters, 764 How Insects Lost Their Abdominal Limbs, 766 Modification of Flight Limbs Might Arise from the Evolution of Regulatory DNA Sequences, 767 GENOME EVOLUTION AND HUMAN ORIGINS, 769 Diverse Animals Contain Remarkably Similar Sets of Genes, 769 Many Animals Contain Anomalous Genes, 769 Synteny Is Evolutionarily Ancient, 770 Deep Sequencing Is Being Used to Explore Human Origins, 772 SUMMARY, 772 BIBLIOGRAPHY, 773 QUESTIONS, 774 22 Systems Biology, 775 REGULATORY CIRCUITS, 776 FEED-FORWARD LOOPS, 784 AUTOREGULATION, 776 Negative Autoregulation Dampens Noise and Allows a Rapid Response Time, 777 Gene Expression Is Noisy, 777 Positive Autoregulation Delays Gene Expression, 779 BISTABILITY, 780 Some Regulatory Circuits Persist in Alternative Stable States, 780 Bimodal Switches Vary in Their Persistence, 781 KEY EXPERIMENTS BOX 22-1 Bistability and Hysteresis, 782 Feed-Forward Loops Are Three-Node Networks with Beneficial Properties, 784 Feed-Forward Loops Are Used in Development, 786 OSCILLATING CIRCUITS, 786 Some Circuits Generate Oscillating Patterns of Gene Expression, 786 Synthetic Circuits Mimic Some of the Features of Natural Regulatory Networks, 789 SUMMARY, 790 BIBLIOGRAPHY, 791 QUESTIONS, 791 PART 6: APPENDICES, 793 APPENDIX 1: Model Organisms, 797 BACTERIOPHAGE, 798 Assays of Phage Growth, 800 The Single-Step Growth Curve, 800 Phage Crosses and Complementation Tests, 801 Transduction and Recombinant DNA, 801 BACTERIA, 802 Assays of Bacterial Growth, 803 Bacteria Exchange DNA by Sexual Conjugation, PhageMediated Transduction, and DNA-Mediated Transformation, 803 Bacterial Plasmids Can Be Used as Cloning Vectors, 805 Transposons Can Be Used to Generate Insertional Mutations and Gene and Operon Fusions, 805 Studies on the Molecular Biology of Bacteria Have Been Enhanced by Recombinant DNA Technology, Detailed Contents Whole-Genome Sequencing, and Transcriptional Profiling, 806 Biochemical Analysis Is Especially Powerful in Simple Cells with Well-Developed Tools of Traditional and Molecular Genetics, 806 Bacteria Are Accessible to Cytological Analysis, 807 Phage and Bacteria Told Us Most of the Fundamentals Things about the Gene, 807 Synthetic Circuits and Regulatory Noise, 808 BAKER’S YEAST, SACCHAROMYCES CEREVISIAE, 808 The Existence of Haploid and Diploid Cells Facilitates Genetic Analysis of S. cerevisiae, 809 Generating Precise Mutations in Yeast Is Easy, 810 S. cerevisiae Has a Small, Well-Characterized Genome, 810 S. cerevisiae Cells Change Shape as They Grow, 810 ARABIDOPSIS, 811 Arabidopsis Has a Fast Life Cycle with Haploid and Diploid Phases, 812 Arabidopsis Is Easily Transformed for Reverse Genetics, 813 Arabidopsis Has a Small Genome That Is Readily Manipulated, 813 Epigenetics, 814 Plants Respond to the Environment, 815 Development and Pattern Formation, 815 APPENDIX 2: Answers, 831 Chapter 1, 831 Chapter 2, 831 Chapter 3, 832 Chapter 4, 833 Chapter 5, 833 Chapter 6, 834 Chapter 7, 834 Chapter 8, 835 Chapter 9, 835 Chapter 10, 836 Chapter 11, 837 Index, 845 Chapter 12, 837 Chapter 13, 838 Chapter 14, 839 Chapter 15, 839 Chapter 16, 840 Chapter 17, 841 Chapter 18, 841 Chapter 19, 843 Chapter 20, 843 Chapter 21, 843 Chapter 22, 844 xxxi THE NEMATODE WORM, CAENORHABDITIS ELEGANS, 816 C. elegans Has a Very Rapid Life Cycle, 816 C. elegans Is Composed of Relatively Few, Well-Studied Cell Lineages, 817 The Cell Death Pathway Was Discovered in C. elegans, 818 RNAi Was Discovered in C. elegans, 818 THE FRUIT FLY, DROSOPHILA MELANOGASTER, 819 Drosophila Has a Rapid Life Cycle, 819 The First Genome Maps Were Produced in Drosophila, 820 Genetic Mosaics Permit the Analysis of Lethal Genes in Adult Flies, 822 The Yeast FLP Recombinase Permits the Efficient Production of Genetic Mosaics, 823 It Is Easy to Create Transgenic Fruit Flies that Carry Foreign DNA, 824 THE HOUSE MOUSE, MUS MUSCULUS, 825 Mouse Embryonic Development Depends on Stem Cells, 826 It Is Easy to Introduce Foreign DNA into the Mouse Embryo, 827 Homologous Recombination Permits the Selective Ablation of Individual Genes, 827 Mice Exhibit Epigenetic Inheritance, 829 BIBLIOGRAPHY, 830 This page intentionally left blank Box Contents ADVANCED CONCEPTS BOX 1-1 Mendelian Laws, 6 BOX 3-1 The Uniqueness of Molecular Shapes and the Concept of Selective Stickiness, 61 BOX 6-1 Ramachandran Plot: Permitted Combinations of Main-Chain Torsion Angles f and c, 124 BOX 6-2 Glossary of Terms, 130 BOX 6-3 The Antibody Molecule as an Illustration of Protein Domains, 133 BOX 9-3 ATP Control of Protein Function: Loading a Sliding Clamp, 282 BOX 9-5 E. coli DNA Replication Is Regulated by DnaA.ATP Levels and SeqA, 294 BOX 10-3 Quantitation of DNA Damage and Its Effects on Cellular Survival and Mutagenesis, 323 BOX 10-6 The Y Family of DNA Polymerases, 336 BOX 11-1 How to Resolve a Recombination Intermediate with Two Holliday Junctions, 350 BOX 12-2 The Xer Recombinase Catalyzes the Monomerization of Bacterial Chromosomes and of Many Bacterial Plasmids, 392 BOX 12-4 Mechanism of Transposition Target Immunity, 413 BOX 13-2 The Single-Subunit RNA Polymerases, 443 BOX 15-1 CCA-Adding Enzymes: Synthesizing RNA without a Template, 513 BOX 15-2 Selenocysteine, 520 BOX 15-3 uORFs and IRESs: Exceptions That Prove the Rule, 533 BOX 15-4 GTP-Binding Proteins, Conformational Switching, BOX 16-1 BOX 18-4 BOX 19-5 BOX 20-1 BOX 21-2 BOX 21-3 BOX 21-6 BOX 21-8 and the Fidelity and Ordering of the Events of Translation, 546 Expanding the Genetic Code, 589 Concentration, Affinity, and Cooperative Binding, 641 Is There a Histone Code?, 691 Amino Acid Biosynthetic Operons Are Controlled by Attenuation, 707 Review of Cytoskeleton: Asymmetry and Growth, 741 Overview of Drosophila Development, 748 Gradient Thresholds, 757 Homeotic Genes of Drosophila Are Organized in Special Chromosome Clusters, 764 KEY EXPERIMENTS BOX 1-2 Genes Are Linked to Chromosomes, 10 BOX 2-1 Chargaff’s Rules, 26 BOX 2-2 Evidence That Genes Control Amino Acid Sequences in Proteins, 31 BOX 4-1 DNA Has 10.5 bp per Turn of the Helix in Solution: The Mica Experiment, 84 BOX 4-2 How Spots on an X-Ray Film Reveal the Structure of DNA, 88 BOX 4-3 Proving that DNA Has a Helical Periodicity of 10.5 bp per Turn from the Topological Properties of DNA Rings, 103 BOX 6-4 Three-Dimensional Structure of a Protein Is Specified by Its Amino Acid Sequence (Anfinsen Experiment), 135 BOX 7-2 Sequenators Are Used for High-Throughput Sequencing, 163 BOX 8-1 Micrococcal Nuclease and the DNA Associated with the Nucleosome, 226 BOX 8-2 Nucleosomes and Superhelical Density, 230 BOX 8-3 Determining Nucleosome Position in the Cell, 245 BOX 9-4 The Identification of Origins of Replication and Replicators, 290 BOX 12-3 Maize Elements and the Discovery of Transposons, 408 BOX 14-1 Adenovirus and the Discovery of Splicing, 471 BOX 14-2 Converting Group I Introns into Ribozymes, 479 BOX 14-3 Identification of Docking Site and Selector Sequences, 490 BOX 18-1 Activator Bypass Experiments, 624 BOX 18-2 Jacob, Monod, and the Ideas behind Gene Regulation, 628 BOX 18-5 Evolution of the l Switch, 645 BOX 18-6 Genetic Approaches That Identified Genes Involved in the Lytic/Lysogenic Choice, 649 xxxiii xxxiv Box Contents BOX 19-4 Evolution of a Regulatory Circuit, 683 BOX 20-2 Discovery of miRNAs and RNAi, 722 BOX 21-4 Activator Synergy, 752 BOX 21-7 cis-Regulatory Sequences in Animal Development and Evolution, 759 BOX 22-1 Bistability and Hysteresis, 782 MEDICAL CONNECTIONS BOX 5-1 An RNA Switch Controls Protein Synthesis by Murine Leukemia Virus, 112 BOX 9-2 Anticancer and Antiviral Agents Target DNA Replication, 268 BOX 9-6 Aging, Cancer, and the Telomere Hypothesis, 307 BOX 10-1 Expansion of Triple Repeats Causes Disease, 316 BOX 10-2 The Ames Test, 321 BOX 10-4 Linking Nucleotide Excision Repair and Translesion Synthesis to a Genetic Disorder in Humans, 330 BOX 10-5 Nonhomologous End Joining, 332 BOX 11-2 The Product of the Tumor Suppressor Gene BRCA2 Interacts with Rad51 Protein and Controls Genome Stability, 367 BOX 11-3 Proteins Associated with Premature Aging and Cancer Promote an Alternative Pathway for Holliday Junction Processing, 368 BOX 12-1 Application of Site-Specific Recombination to Genetic Engineering, 386 BOX 14-4 Defects in Pre-mRNA Splicing Cause Human Disease, 497 BOX 14-5 Deaminases and HIV, 503 BOX 15-5 Antibiotics Arrest Cell Division by Blocking Specific Steps in Translation, 552 BOX 15-7 A Frontline Drug in Tuberculosis Therapy Targets SsrA Tagging, 565 BOX 18-3 Blocking Virulence by Silencing Pathways of Intercellular Communication, 635 BOX 19-3 Histone Modifications, Transcription Elongation, and Leukemia, 670 BOX 19-6 Transcriptional Repression and Human Disease, 696 BOX 20-3 microRNAs and Human Disease, 727 BOX 21-1 Formation of iPS Cells, 734 BOX 21-5 Stem Cell Niche, 755 TECHNIQUES BOX 5-2 Creating an RNA Mimetic of the Green Fluorescent Protein by Directed Evolution, 115 BOX 7-1 Forensics and the Polymerase Chain Reaction, 160 BOX 9-1 Incorporation Assays Can Be Used to Measure Nucleic Acid and Protein Synthesis, 261 BOX 13-1 BOX 15-6 BOX 19-1 BOX 19-2 Consensus Sequences, 436 Ribosome and Polysome Profiling, 561 The Two-Hybrid Assay, 664 The ChIP-Chip and ChIP-Seq Assays Are the Best Method for Identifying Enhancers, 666 P A R T 1 HISTORY O U T L I N E CHAPTER 1 The Mendelian View of the World, 5 † CHAPTER 2 Nucleic Acids Convey Genetic Information, 21 2 Part 1 U NLIKE THE REST OF THIS BOOK, the two chapters that make up Part 1 contain material largely unchanged from earlier editions. We nevertheless keep these chapters because the material remains as important as ever. Specifically, Chapters 1 and 2 provide an historical account of how the field of genetics and the molecular basis of genetics were established. Key ideas and experiments are described. Chapter 1 addresses the founding events in the history of genetics. We discuss everything from Mendel’s famous experiments on peas, which uncovered the basic laws of heredity, to the one gene encodes one enzyme hypothesis of Garrod. Chapter 2 describes the revolutionary development of molecular biology that was started with Avery’s discovery that DNA was the genetic material, and continued with James Watson and Francis Crick’s proposal that the structure of DNA is a double helix, and the elucidation of the genetic code and the “central dogma” (DNA “makes” RNA which “makes” protein). Chapter 2 concludes with a discussion of recent developments stemming from the complete sequencing of the genomes of many organisms and the impact this sequencing has on modern biology. PHOTOS FROM THE COLD SPRING HARBOR LABORATORY ARCHIVES Vernon Ingram, Marshall W. Nirenberg, and Matthias Staehelin, 1963 Symposium on Synthesis and Structure of Macromolecules. Ingram demonstrated that genes control the amino acid sequence of proteins; the mutation causing sickle-cell anemia produces a single amino acid change in the hemoglobin protein (Chapter 2). Nirenberg was key in unraveling the genetic code, using protein synthesis directed by artificial RNA templates in vitro (Chapters 2 and 16). For this achievement, he shared in the 1968 Nobel Prize in Physiology or Medicine. Staehelin worked on the small RNA molecules, tRNAs, which translate the genetic code into amino acid sequences of proteins (Chapters 2 and 16). Melvin Calvin, Francis Crick, George Gamow, and James Watson, 1963 Symposium on Synthesis and Structure of Macromolecules. Calvin won the 1961 Nobel Prize in Chemistry for his work on CO2 assimilation by plants. For their proposed structure of DNA, Crick and Watson shared in the 1962 Nobel Prize in Physiology or Medicine (Chapters 2 and 4). Gamow, a physicist attracted to the problem of the genetic code (Chapters 2 and 16), founded an informal group of like-minded scientists called the RNA Tie Club. (He is wearing the club tie, which he designed, in this picture.) History Raymond Appleyard, George Bowen, and Martha Chase, 1953 Symposium on Viruses. Appleyard and Bowen, both phage geneticists, are here shown with Chase, who, in 1952, together with Alfred Hershey, did the simple experiment that finally convinced most people that the genetic material is DNA (Chapter 2). Sydney Brenner and James Watson, 1975 Symposium on The Synapse. Brenner, shown here with Watson, contributed to the discoveries of mRNA and the nature of the genetic code (Chapters 2 and 16); his share of a Nobel Prize, in 2002, however, was for establishing the worm, Caenorhabditis elegans, as a model system for the study of developmental biology (Appendix 1). Max Perutz, 1971 Symposium on Structure and Function of Proteins at the Three-Dimensional Level. Perutz shared, with John Kendrew, the 1962 Nobel Prize for Chemistry; using X-ray crystallography, and after 25 years of effort, they were the first to solve the atomic structures of proteins—hemoglobin and myoglobin, respectively (Chapter 6). Francis Crick, 1963 Symposium on Synthesis and Structure of Macromolecules. In addition to his role in solving the structure of DNA, Crick was an intellectual driving force in the development of molecular biology during the field’s critical early years. His “adaptor hypothesis” ( published in the RNA Tie Club newsletter) predicted the existence of molecules required to translate the genetic code of RNA into the amino acid sequence of proteins. Only later were tRNAs found to do just that (Chapter 15). 3 4 Part 1 Seymour Benzer, 1975 Symposium on The Synapse. Using phage genetics, Benzer defined the smallest unit of mutation, which turned out later to be a single nucleotide (Chapter 1 and Appendix 1). This same work also provided an experimental definition of the gene—which he called a cistron—using functional complementation tests. Later, his studies focused on behavior, using the fruit fly as a model. Charles Yanofsky, 1966 Symposium on The Genetic Code. Yanofsky (right), together with Sydney Brenner, proved colinearity of the gene—that is, that successive groups of nucleotides encoded successive amino acids in the protein product (Chapter 2). He later discovered the first example of transcriptional regulation by RNA structure in his detailed analysis of attenuation at the tryptophan operon of Escherichia coli (Chapter 20). He is pictured here talking to Michael Chamberlin, who studied transcription initiation by RNA polymerase. Calvin Bridges, 1934 Symposium on Aspects of Growth. Bridges (shown reading the newspaper) was part of T.H. Morgan’s famous “fly group” that pioneered the development of the fruit fly Drosophila as a model genetic organism (Chapter 1 and Appendix 1). With him is John T. Buchholtz, a plant geneticist who was a summer visitor at CSHL at the time, and who, in 1941, became President of the Botanical Society of America. Edwin Chargaff, 1947 Symposium on Nucleic Acids and Nucleoproteins. The eminent nucleic acid biochemist Chargaff’s famous ratios—that the amount of adenine in a DNA sample matched that of thymine, and the amount of cytosine matched that of guanine— were later understood in the context of Watson and Crick’s DNA double helix structure. Perhaps frustrated that he had never come up with base pairs himself, he became a bitter critic of molecular biology, an occupation he described as “essentially the practice of biochemistry without a license.” C H A P T E R 1 Aa AA aa Aa The Mendelian View of the World T IS EASY TO CONSIDER HUMAN BEINGS UNIQUE among living organisms. We alone have developed complicated languages that allow meaningful and complex interplay of ideas and emotions. Great civilizations have developed and changed our world’s environment in ways inconceivable for any other form of life. There has always been a tendency, therefore, to think that something special differentiates humans from every other species. This belief has found expression in the many forms of religion through which we seek the origin of and explore the reasons for our existence and, in so doing, try to create workable rules for conducting our lives. Little more than a century ago, it seemed natural to think that, just as every human life begins and ends at a ﬁxed time, the human species and all other forms of life must also have been created at a ﬁxed moment. This belief was ﬁrst seriously questioned almost 150 years ago, when Charles Darwin and Alfred R. Wallace proposed their theories of evolution, based on the selection of the most ﬁt. They stated that the various forms of life are not constant but continually give rise to slightly different animals and plants, some of which adapt to survive and multiply more effectively. At the time of this theory, they did not know the origin of this continuous variation, but they did correctly realize that these new characteristics must persist in the progeny if such variations are to form the basis of evolution. At ﬁrst, there was a great furor against Darwin, most of it coming from people who did not like to believe that humans and the rather obscene-looking apes could have a common ancestor, even if this ancestor had lived some 10 million years ago. There was also initial opposition from many biologists who failed to ﬁnd Darwin’s evidence convincing. Among these was the famous naturalist Jean L. Agassiz, then at Harvard, who spent many years writing against Darwin and Darwin’s champion, Thomas H. Huxley, the most successful of the popularizers of evolution. But by the end of the 19th century, the scientiﬁc argument was almost complete; both the current geographic distribution of plants and animals and their selective occurrence in the fossil records of the geologic past were explicable only by postulating that continuously evolving groups of organisms had descended from a common ancestor. Today, evolution is an accepted fact for everyone except a fundamentalist minority, whose objections are based not on reasoning but on doctrinaire adherence to religious principles. An immediate consequence of Darwinian theory is the realization that life ﬁrst existed on our Earth more than 4 billion years ago in a simple I 5 O U T L I N E Mendel’s Discoveries, 6 † Chromosomal Theory of Heredity, 8 † Gene Linkage and Crossing Over, 9 † Chromosome Mapping, 11 † The Origin of Genetic Variability through Mutations, 13 † Early Speculations about What Genes Are and How They Act, 15 † Preliminary Attempts to Find a Gene–Protein Relationship, 16 † Visit Web Content for Structural Tutorials and Interactive Animations 6 Chapter 1 form, possibly resembling the bacteria—the simplest variety of life known today. The existence of such small bacteria tells us that the essence of the living state is found in very small organisms. Evolutionary theory further suggests that the basic principles of life apply to all living forms. MENDEL’S DISCOVERIES Gregor Mendel’s experiments traced the results of breeding experiments (genetic crosses) between strains of peas differing in well-deﬁned characteristics, like seed shape (round or wrinkled), seed color (yellow or green), pod shape (inﬂated or wrinkled), and stem length (long or short). His concentration on well-deﬁned differences was of great importance; many breeders had previously tried to follow the inheritance of more gross qualities, like body weight, and were unable to discover any simple rules about their transmission from parents to offspring (see Box 1-1, Mendelian Laws). The Principle of Independent Segregation After ascertaining that each type of parental strain bred true—that is, produced progeny with particular qualities identical to those of the parents— Mendel performed a number of crosses between parents (P) differing in single characteristics (such as seed shape or seed color). All the progeny (F1 ¼ ﬁrst ﬁlial generation) had the appearance of one parent only. For example, in a cross between peas having yellow seeds and peas having green seeds, all the progeny had yellow seeds. The trait that appears in the F1 progeny is called dominant, whereas the trait that does not appear in Fl is called recessive. } A D VA N C E D C O N C E P T S B O X 1-1 Mendelian Laws The most striking attribute of a living cell is its ability to transmit hereditary properties from one cell generation to another. The existence of heredity must have been noticed by early humans, who witnessed the passing of characteristics, like eye or hair color, from parents to offspring. Its physical basis, however, was not understood until the ﬁrst years of the 20th century, when, during a remarkable period of creative activity, the chromosomal theory of heredity was established. Hereditary transmission through the sperm and egg became known by 1860, and in 1868 Ernst Haeckel, noting that sperm consists largely of nuclear material, postulated that the nucleus is responsible for heredity. Almost 20 years passed before the chromosomes were singled out as the active factors, because the details of mitosis, meiosis, and fertilization had to be worked out ﬁrst. When this was accomplished, it could be seen that, unlike other cellular constituents, the chromosomes are equally divided between daughter cells. Moreover, the complicated chromosomal changes that reduce the sperm and egg chromosome number to the haploid number during meiosis became understandable as nec- essary for keeping the chromosome number constant. These facts, however, merely suggested that chromosomes carry heredity. Proof came at the turn of the century with the discovery of the basic rules of heredity. The concepts were ﬁrst proposed by Gregor Mendel in 1865 in a paper entitled “Experiments in Plant Hybridization” given to the Natural Science Society at Brno. In his presentation, Mendel described in great detail the patterns of transmission of traits in pea plants, his conclusions of the principles of heredity, and their relevance to the controversial theories of evolution. The climate of scientiﬁc opinion, however, was not favorable, and these ideas were completely ignored, despite some early efforts on Mendel’s part to interest the prominent biologists of his time. In 1900, 16 years after Mendel’s death, three plant breeders working independently on different systems conﬁrmed the signiﬁcance of Mendel’s forgotten work. Hugo de Vries, Karl Correns, and Erich von Tschermak-Seysenegg, all doing experiments related to Mendel’s, reached similar conclusions before they knew of Mendel’s work. The Mendelian View of the World The meaning of these results became clear when Mendel set up genetic crosses between F1 offspring. These crosses gave the important result that the recessive trait reappeared in approximately 25% of the F2 progeny, whereas the dominant trait appeared in 75% of these offspring. For each of the seven traits he followed, the ratio in F2 of dominant to recessive traits was always approximately 3:1. When these experiments were carried to a third (F3) progeny generation, all the F2 peas with recessive traits bred true ( produced progeny with the recessive traits). Those with dominant traits fell into two groups: one third bred true ( produced only progeny with the dominant trait); the remaining two-thirds again produced mixed progeny in a 3:1 ratio of dominant to recessive. Mendel correctly interpreted his results as follows (Fig. 1-1): the various traits are controlled by pairs of factors (which we now call genes), one factor derived from the male parent, the other from the female. For example, pure-breeding strains of round peas contain two versions (or alleles) of the roundness gene (RR), whereas pure-breeding wrinkled strains have two copies of the wrinkledness (rr) allele. The round-strain gametes each have one gene for roundness (R); the wrinkled-strain gametes each have one gene for wrinkledness (r). In a cross between RR and rr, fertilization produces an Fl plant with both alleles (Rr). The seeds look round because R is dominant over r. We refer to the appearance or physical structure of an individual as its phenotype, and to its genetic composition as its genotype. Individuals with identical phenotypes may possess different genotypes; thus, to determine the genotype of an organism, it is frequently necessary to perform genetic crosses for several generations. The term homozygous refers to a gene pair in which both the maternal and paternal genes are identical (e.g., RR or rr). In contrast, those gene pairs in which paternal and maternal genes are different (e.g., Rr) are called heterozygous. One or several letters or symbols may be used to represent a particular gene. The dominant allele of the gene may be indicated by a capital letter (R), by a superscript þ (r þ), or by a þ standing alone. In our discussions here, we use the ﬁrst convention in which the dominant allele is represented by a capital letter and the recessive allele by the lowercase letter. It is important to notice that a given gamete contains only one of the two copies (one allele) of the genes present in the organism it comes from (e.g., either R or r, but never both) and that the two types of gametes are produced in equal numbers. Thus, there is a 50:50 chance that a given gamete from an Fl pea will contain a particular gene (R or r). This choice is purely random. We do not expect to ﬁnd exact 3:1 ratios when we examine a limited number of F2 progeny. The ratio will sometimes be slightly higher and other times slightly lower. But as we look at increasingly larger samples, we expect that the ratio of peas with the dominant trait to peas with the recessive trait will approximate the 3:1 ratio more and more closely. The reappearance of the recessive characteristic in the F2 generation indicates that recessive alleles are neither modiﬁed nor lost in the Fl (Rr) generation, but that the dominant and recessive genes are independently transmitted and so are able to segregate independently during the formation of sex cells. This principle of independent segregation is frequently referred to as Mendel’s ﬁrst law. Some Alleles Are neither Dominant nor Recessive In the crosses reported by Mendel, one member of each gene pair was clearly dominant to the other. Such behavior, however, is not universal. Sometimes the heterozygous phenotype is intermediate between the two homozygous 7 parental generation RR rr R r gametes hybrid F1 generation Rr