Genetics and Molecular Biology - Johns Hopkins University

1 Genetics and Molecular BiologyGenetics andMolecular BiologyS E C O N D E D I T I O N Robert SchleifDepartment of BiologyThe Johns Hopkins UniversityBaltimore, MarylandThe Johns Hopkins University Press Baltimore and London 1986 by Addison-Wesley Publishing Company 1993 by Robert SchleifAll rights reservedPrinted in the United States of America on acid-free paperThe Johns Hopkins University Press2715 North Charles StreetBaltimore, Maryland 21218-4319 The Johns Hopkins Press Ltd., LondonLibrary of Congress Cataloging-in-Publication DataSchleif, Robert F. Genetics and Molecular Biology / by Robert Schleif. 2nd ed. p. cm. Includes bibliographical references and index. ISBN 0-8018-4673-0 (acid-free paper). ISBN 0-8018-4674-9 (pbk : acid-freepaper) 1. Molecular Genetics . I. 1993 The catalog record for this book is available from the British book evolved from a course in Molecular Biology which I have beenteaching primarily to graduate students for the past twenty the subject is now mature, it is possible to present the materialby covering the principles and encouraging students to learn how toapply them.

2 Such an approach is particularly efficient as the subject ofmolecular Genetics now is far too advanced, large, and complex formuch value to come from attempting to cover the material in anencyclopedia-like fashion or teaching the definitions of the relevantwords in a dictionary-like approach. Only the core of Molecular geneticscan be covered by the present approach. Most of the remainder of thevast subject however, is a logical extension of the ideas and principlespresented here. One consequence of the principles and analysis ap-proach taken here is that the material is not easy. Thinking and learningto reason from the fundamentals require serious effort, but ultimately,are more efficient and more rewarding than mere auxiliary objective of this presentation is to help students developan appreciation for elegant and beautiful experiments. A substantialnumber of such experiments are explained in the text, and the citedpapers contain many book contains three types of information.

3 The main part of eachchapter is the text. Following each chapter are references and are arranged by topic, and one topic is Suggested Read-ings . The additional references cited permit a student or researcher tofind many of the fundamental papers on a topic. Some of these are ontopics not directly covered in the text. Because solving problems helpsfocus one s attention and stimulates understanding, many thought-pro-voking problems or paradoxes are provided. Some of these require useof material in addition to the text. Solutions are provided to about halfof the the ideal preparation for taking the course and using thebook would be the completion of preliminary courses in biochemistry, Molecular Biology , cell Biology , and physical chemistry, few studentshave such a background. Most commonly, only one or two of theabove-mentioned courses have been taken, with some students comingfrom a more physical or chemical background, and other studentscoming from a more biological course consists of two lectures and one discussion session perweek, with most chapters being covered in one lecture.

4 The lecturesoften summarize material of a chapter and then discuss in depth arecent paper that extends the material of the chapter. Additional read-ings of original research papers are an important part of the course forgraduate students, and typically such two papers are assigned perlecture. Normally, two problems from the ends of the chapters areassigned per lecture. Many of the ideas presented in the book have been sharpened by myfrequent discussions with Pieter Wensink, and I thank him for this. Ithank my editors, James Funston for guidance on the first edition andYale Altman and Richard O Grady for ensuring the viability of thesecond edition. I also thank members of my laboratory and the followingwho read and commented on portions of the manuscript: KarenBeemon, Howard Berg, Don Brown, Victor Corces, Jeff Corden, DavidDraper, Mike Edidin, Bert Ely, Richard Gourse, Ed Hedgecock, RogerHendrix, Jay Hirsh, Andy Hoyt, Amar Klar, Ed Lattman, RogerMcMacken, Howard Nash.

5 And Peter PrefaceContents1 An Overview of Cell Structure and Function1 Cell s Need for Immense Amounts of Information2 Rudiments of Prokaryotic Cell Structure2 Rudiments of Eukaryotic Cell Structure5 Packing DNA into Cells7 Moving Molecules into or out of Cells8 Diffusion within the Small Volume of a Cell13 Exponentially Growing Populations14 Composition Change in Growing Cells15 Age Distribution in Populations of Growing Cells15 Problems16 References182 Nucleic Acid and Chromosome Structure21 The Regular Backbone Of DNA22 Grooves in DNA and Helical Forms of DNA23 Dissociation and Reassociation of Base-paired Strands26 Reading Sequence Without Dissociating Strands27 Electrophoretic Fragment Separation28 Bent DNA Sequences29 Measurement of Helical Pitch31 Topological Considerations in DNA Structure32 Generating DNA with Superhelical Turns33 Measuring Superhelical Turns34 Determining Lk, Tw, and Wr in Hypothetical Structures 36 Altering Linking Number37 Biological Significance of Superhelical Turns39viiThe Linking Number Paradox of Nucleosomes40 General Chromosome Structure41 Southern Transfers to Locate Nucleosomes on Genes41 ARS Elements, Centromeres, and Telomeres43 Problems 44 References473 DNA Synthesis53A.

6 Enzymology54 Proofreading, Okazaki Fragments, and DNA Ligase54 Detection and Basic Properties of DNA Polymerases57In vitro DNA Replication60 Error and Damage Correction 62B. Physiological Aspects66 DNA Replication Areas In Chromosomes 66 Bidirectional Replication from E. coli Origins 67 The DNA Elongation Rate69 Constancy of the E. coli DNA Elongation Rate71 Regulating Initiations 72 Gel Electrophoresis Assay of Eukaryotic Replication Origins74 How Fast Could DNA Be Replicated?76 Problems 78 References794 RNA Polymerase and RNA Initiation85 Measuring the Activity of RNA Polymerase 86 Concentration of Free RNA Polymerase in Cells89 The RNA Polymerase in Escherichia coli90 Three RNA Polymerases in Eukaryotic Cells91 Multiple but Related Subunits in Polymerases92 Multiple Sigma Subunits95 The Structure of Promoters96 Enhancers 99 Enhancer-Binding Proteins100 DNA Looping in Regulating Promoter Activities102 Steps of the Initiation Process104 Measurement of Binding and Initiation Rates 105 Relating Abortive Initiations to Binding and Initiating107 Roles of Auxiliary Transcription Factors109 Melted DNA Under RNA Polymerase110 Problems

7 111 References1135 Transcription, Termination, and RNA Processing119 Polymerase Elongation Rate119viii ContentsTranscription Termination at Specific Sites 121 Termination122 Processing Prokaryotic RNAs After Synthesis 125S1 Mapping to Locate 5 and 3 Ends of Transcripts126 Caps, Splices, Edits, and Poly-A Tails on Eukaryotic RNAs127 The Discovery and Assay of RNA Splicing 128 Involvement of the U1 snRNP Particle in Splicing131 Splicing Reactions and Complexes134 The Discovery of Self-Splicing RNAs135A Common Mechanism for Splicing Reactions137 Other RNA Processing Reactions139 Problems 140 References1426 Protein Structure149 The Amino Acids150 The Peptide Bond 153 Electrostatic Forces that Determine Protein Structure154 Hydrogen Bonds and the Chelate Effect158 Hydrophobic Forces159 Thermodynamic Considerations of Protein Structure161 Structures within Proteins162 The Alpha Helix, Beta Sheet.

8 And Beta Turn164 Calculation of Protein Tertiary Structure166 Secondary Structure Predictions168 Structures of DNA-Binding Proteins170 Salt Effects on Protein-DNA Interactions173 Locating Specific Residue-Base Interactions174 Problems175 References1777 Protein Synthesis183A. chemical Aspects184 Activation of Amino Acids During Protein Synthesis184 Fidelity of Aminoacylation185 How Synthetases Identify the Correct tRNA Molecule187 Decoding the Message188 Base Pairing between Ribosomal RNA and Messenger191 Experimental Support for the Shine-Dalgarno Hypothesis192 Eukaryotic Translation and the First AUG194 Tricking the Translation Machinery into Initiating195 Protein Elongation197 Peptide Bond Formation198 Translocation198 Termination, Nonsense, and Suppression199 Chaperones and Catalyzed Protein Folding202 Contents ixResolution of a Paradox202B. Physiological Aspects203 Messenger Instability203 Protein Elongation Rates204 Directing Proteins to Specific Cellular Sites207 Verifying the Signal Peptide Model208 The Signal Recognition Particle and Translocation210 Expectations for Ribosome Regulation211 Proportionality of Ribosome Levels and Growth Rates212 Regulation of Ribosome Synthesis214 Balancing Synthesis of Ribosomal Components216 Problems218 References2208 Genetics227 Mutations227 Point Mutations, Deletions, Insertions, and Damage228 Classical Genetics of Chromosomes231 Complementation, Cis, Trans, Dominant, and Recessive233 Mechanism of a trans Dominant Negative Mutation234 genetic Recombination235 Mapping by Recombination Frequencies236 Mapping by Deletions239 Heteroduplexes and genetic Recombination239 Branch Migration and Isomerization241 Elements of Recombination in E.

9 Coli, RecA, RecBCD, and Chi243 genetic Systems244 Growing Cells for Genetics Experiments 245 Testing Purified Cultures, Scoring 246 Isolating Auxotrophs, Use of Mutagens and Replica Plating247 genetic Selections 248 Mapping with Generalized Transducing Phage 250 Principles of Bacterial Sex 251 Elements of Yeast Genetics 253 Elements of Drosophila Genetics254 Isolating Mutations in Muscle or Nerve in Drosophila 255 Fate Mapping and Study of Tissue-Specific Gene Expression256 Problems257 References2619 genetic Engineering and Recombinant DNA265 The Isolation of DNA266 The Biology of Restriction Enzymes268 Cutting DNA with Restriction Enzymes271 Isolation of DNA Fragments272x ContentsJoining DNA Fragments272 Vectors: Selection and Autonomous DNA Replication274 Plasmid Vectors274A Phage Vector for Bacteria278 Vectors for Higher Cells279 Putting DNA Back into Cells281 Cloning from RNA282 Plaque and Colony Hybridization for Clone Identification283 Walking Along a Chromosome to Clone a Gene284 Arrest of Translation to Assay for DNA of a Gene285 chemical DNA Sequencing286 Enzymatic DNA Sequencing289 Problems291 References29310 Advanced genetic Engineering297 Finding Clones from a Known Amino Acid Sequence297 Finding Clones Using Antibodies Against a Protein298 Southern, Northern, and Western Transfers300 Polymerase Chain Reaction302 Isolation of Rare Sequences Utilizing PCR305 Physical and genetic Maps of Chromosomes 306 Chromosome Mapping307 DNA Fingerprinting Forensics310 Megabase Sequencing311 Footprinting, Premodification and Missing Contact Probing313 Antisense RNA.

10 Selective Gene Inactivation317 Hypersynthesis of Proteins317 Altering Cloned DNA by in vitro Mutagenesis318 mutagenesis with Chemically Synthesized DNA321 Problems323 References32511 Repression and the lac Operon331 Background of the lac Operon332 The Role of Inducer Analogs in the Study of the lac Operon334 Proving lac Repressor is a Protein335An Assay for lac Repressor336 The Difficulty of Detecting Wild-Type lac Repressor338 Detection and Purification of lac Repressor340 Repressor Binds to DNA: The Operator is DNA341 The Migration Retardation Assay and DNA Looping343 The Isolation and Structure of Operator344In vivo Affinity of Repressor for Operator346 The DNA-binding Domain of lac Repressor346A Mechanism for Induction348 Contents xiProblems349 References35312 Induction, Repression, and the araBAD Operon359 The Sugar Arabinose and Arabinose Metabolism360 Genetics of the Arabinose System362 Detection and Isolation of AraC Protein364 Repression by AraC366 Regulating AraC Synthesis368 Binding Sites of the ara Regulatory Proteins369 DNA Looping and Repression of araBAD371In vivo Footprinting Demonstration of Looping373 How AraC Protein Loops and Unloops373 Why Looping is Biologically Sensible376 Why Positive Regulators are a Good Idea376 Problems377 References37913 Attenuation and the trp Operon385 The Aromatic Amino Acid Synthetic Pathway and its Regulation386 Rapid Induction Capabilities of the trp Operon388 The Serendipitous Discovery of trp Enzyme Hypersynthesis390 Early Explorations of the Hypersynthesis392trp Multiple Secondary Structures in trp Leader RNA396 Coupling Translation to Termination397 RNA Secondary Structure and the Attenuation Mechanism399 Other