FOURTH EDITION
`
`MOLECULAR
`CELIL
`BIOLOGY
`
`Harvey todish
`
`.Arnold Berk
`
`S. Lawrence Zipursky
`
`Paul Matsudaira
`
`David Baltimore
`
`James Darnell
`
`Media Connected :
`II W. H. FREEMAN AND COMPA~
`
`I
`
`Alnylam Exh. 1059
`
`

`

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`Library of Congress Cataloging-in-Publication Data
`
`r, _1 j
`,,
`' , ·•
`
`)
`
`A
`
`Molecular cell biology /-Harvey Lodish p [et al.] - 4th ed.
`p. cm.
`Includes bibliographical references.
`ISBN 0-7167-3136-3
`1. Cytology. 2. Molecular biology.
`QH581.2.M655
`1999
`571.6-dc21
`
`I. Lodish, Harvey F.
`
`99-30831
`CIP
`
`© 1986, 1990, 1995, 2000 by W. H . . Freeman and Company. All rights reserved.
`
`No part of this book may be reproduced by any mechanical, photographic, or
`electronic process, or in the form of a phonographic recording, nor may it be
`stored .in a retrieval system, transmitted, or otherwise copied for public or private
`use, without written permission from the publisher.
`
`Printed in the United States of America
`
`W. H. Freeman and Company
`41 Madison Avenue, New York, New York 10010
`Houndsmills, Basingstoke RG21 6XS, England
`
`Second printing, 2000
`
`

`

`• IA
`
`PART I Laying the Groundwork
`
`PART Ill Building and Fueling
`the Cell
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`The Dynamic Cell 1
`
`Chemical Foundations 14
`
`Protein Structure and Function 50
`
`15 Transport across Cell Membranes 578
`
`16 Cellular: Energetics: Glycolysis, Aerobic
`Oxidation, apd Photosynthesis 616
`
`Nucleic Acids, the Genetic Code, and the
`Synthesis of Macromolecules 100
`
`17
`
`Protein Sorting: Organelle Biogenesis and
`Protein Secretion 675
`
`Biomembranes and the Subcellular
`Organization of Eukaryotic Cells 138
`
`Manipulating Cells and Viruses in
`Culture 180
`
`Recombinant DNA and Genomics 207
`
`Genetic Analysis in Cell Biology 254
`
`18 Cell Motility and Shape I:
`Microfilaments 751
`
`19 Cell Motility and Shape II: Microtubules
`and Intermediate Filaments 795
`
`PART IV Cell Interactions
`
`PART II Nuclear Control of
`Cellular Activity
`
`20
`
`I,
`Cell-to-Cell Sighaling: Hormones and
`Receptors 848.' · '
`
`I
`
`JI
`
`•
`
`21 Nerve Cells 911
`
`22
`
`Integrating Cells into Tissues 968
`
`23 Cell Interactions in Development 1003
`
`24 Cancer 1054
`
`9 Molecular Structure of Genes and
`Chromosomes 294
`
`10 ReguJation of Transcription Initiation 341
`
`11 RNA Processing, Nuclear Transport, and
`Post-Transcriptional Control 404
`
`12 DNA Repjjcation, Repaii; and
`Recombination 453
`
`13 Regulation of the Eukaryotic Cell Cycle 495
`
`14 Gene Control in Development 537
`
`

`

`eorntents
`1'1m,lr -im1,rl
`
`r
`
`Chapter-Opening Illustrations xxxvii
`
`PART I: Laying the Groundwork
`
`1 The Dynamic Cell
`
`1.1 Evolution: At the Core of Molecular Change 3
`
`1.2 T he Molecules of Life 3
`
`1.3 The Architecture of Cells 5
`Cells Are Surrounded by Water-Impermeable
`Membranes 5
`Membranes Serve Functions Other Than Segregation 6
`Prokaryotes Comprise a Single fylembrane-Limited
`Compartment 7
`Eukaryotic Cells Contain Many Organelles and a
`Complex Cytoskeleton 7
`Cellular DNA Is Packaged within Chromosomes 8
`
`1.4 The Li£~ Cycle of Cells 9
`The Cell Cycle Follows a Regular Timing Mechanism 9
`Mitosis Apportions the Duplicated Chromosomes Equally
`to Daughter Cells 10
`Cell Differentiation Creates New Types of Cells 10
`Cells Die by Suicide 10
`
`1.5 Cells into Tissues 11
`Multicellularity Requires B:i..1:racellular Glues 11
`Tissues Ar.e Organized into Organs 11
`Body Plan and Rudimentary Tissues Form Eady in
`Embryonic Development 12
`1.6 Molecular Cell Biology: An Integrated View of Cells
`at Work 13
`
`MEDIA CONNECTIONS
`Overview: Life Cyclli! of a Cell
`
`, 2 Chemical Foundations
`
`2.1 Covalen t Bonds 15
`Each Atom Can Make a Defined Number of Covalent
`Bonds 16
`The Making or Breaking of Covalent Bonds Involves
`Large Energy Changes 17
`Covalent Bonds Have Characteristic Geometries 17
`Electrons Are Shared Unequally in Polar Covalent
`Bonds 18
`
`Asymmetric Carbon Atoms Are Present ln Most Biological
`Molecules 19
`a and /3 Glycosidic Bonds Link Monosaccharides 21
`2.2 N oncovalent Bonds 22
`The Hydrogen Bond Underlies Water's Chemical and
`Biological Properties 22
`Ionic Interactions Are Attractions between Oppositely
`Charged Ions 23
`Van der Waals Interactions Are Caused by Transient
`Dipoles 24
`Hydrophobic Bonds Cause Nonpolar Molecules to Adhere
`to One Another 25
`Multiple Noncovalent Bonds Cao Confer Binding
`Specificity 26
`Phospholipids Are Amphipathic Molecules 26 •
`The Phospholipid Bilayer Forms the Basic Structure o.f All
`Biomembranes 27
`
`2.3 Chemical Equilibrium 29
`Equilibriwn Constants Reflect the Extent of a Chemical
`Reaction 29
`The Concentration of Complexes Can Be Estimated from
`Equilibrium Constants for Binding Reactions 31
`Biological Fluids Have Characteristic pH Values 31
`Hydrogen Ions Are Released by Acids and Taken Up by
`Bases 32
`The Henderson-HasselbalchJquation Relates pH and Keq
`of an Acid-Base System -' 33
`'1
`Buffers Maintain the pH of _9tracellular and Extracellular
`Fluids 33
`.'
`
`'35
`2.4 Biochemical Energetics
`Living Systems Use Various F·orms of Energy, Which Are
`Interconvertible 35
`The Change in Free Energy 6.G Determines the Direction
`of a Chemical Reaction 36
`The t:i.G of a Reaction Depends on Changes in Enthalpy
`(Bond Energy) and Entropy 36
`Several Parameters Affect the 6.G of a Reaction 37
`' of a Reaction Can Be Calculated from Its K eq 38
`The 6.G0
`Cells Must Expend Energy to Generate Concentration
`Gradients 39
`Many Cellular Processes Involve Oxidation-Reduction
`Reactions 39
`An Unfavorable Chemical Reaction Cao Proceed lf It rs
`Coupled with an EnergeticaUy Favorable Reaction 41
`Hydrolysis of Phosphoanhydride Bonds in ATP Releases
`Substantial Free Energy 41
`ATP ls Used to Fuel Many Cellular Processes 43
`
`

`

`xviii Contents
`
`2.5 Activation Energy arid R~ctiou Rate 45
`Chemical Reactions Proceed through High-Energy
`Transition States 45
`Enzymes Accelerate Biochemical Reactions by Reducing
`Transition-Stare Free Energy 47
`
`MEDIA CONNECTIONS
`Over view: Biologlcal Energy lnterconverslons
`
`3 Protein Structure and Function
`
`3 .1 Hierarchical Structure of Proteins 51
`The Amino Acids Composing Proteins Differ Only in
`Their Side Chains 51
`Peptide Bonds Connect Amino Acids into Linear
`Chains 53
`Four Levels of Structure Determine the Shape of
`Proteins 54
`Graphic Representations of Proteins Highlight Different
`Features 54
`Secondary Structures Are Crucial Elements of Protein
`Architecture 56
`Motifs Are Regular Combinations of Secondary
`Structures 58
`Structural and Functional Domains Are Modules of
`Tertiary Structure 60
`Sequence Homology Suggests Functional and Evolutionary
`Relationships between Proteins 60
`
`3.2 Folding, Modification, and Degradation of
`Proteins 62
`The Information for Protein Foldiog Is Encoded in the
`Sequence 63
`Folding of Proteins in Vivo Is Promoted by
`Chaperones 63
`Chemical Modifications and Processing Alter the
`Biological Activity of Proteins 64
`Cells Degrade Proteins via Several Pathways 66
`Aberrantly Folded Proteins Are Implicated in Slowly
`Developing Diseases 67
`
`3.3 Function al Design of Proteins 68
`Proteins Ate Designed to Bind a Wide Range of
`Molecules 68
`Antibodies Exbibir Precise Ligand-Binding
`Specificity 70
`·
`Enzymes Are Highly Efficient and Specific Catalysts 71
`AJJ Enzyme's Active Site Binds Substrates and Carries Out
`Catalysis 71
`Kinetics of an Enzymatic Reaction Are Described by V max
`and Km 73
`Many Proteins Contain Tightly Bound Prosthetic
`Groups 74
`A Variety of Regulatory Mechanisms Control Protein
`Function 75
`
`3 .4 Membran e Proteins 78
`Proteins Interact with Membranes in Different Ways 78
`Hydrophobic a Heljces in Transmembrane Proteins Ate
`Embedded in the Bilayer 79
`Many Integral Proteins Contain Multiple Transmembrane
`a Helices 79
`Multiple /3 Strands in Porins Form Membrane-Spanning
`"Barrels" 81
`Covalently Attached Hydrocarbon Chains Anchor Some
`Proteins to tbe Membrane 81
`Some Peripheral Proteins Are Soluble En:t.ymes That Act
`on Membrane Components 82.
`3.5 Purifying, Detecting, and Characterizing
`Proteins 83
`Proteins Can Be Removed from Membranes by Detergents
`or High-Salt Solutions 83
`Centrifugation Can Separate J'articles and Molecules That
`Differ in Mass or Density 85
`Electrophoresis Separates Molecules according to Their
`Charge:Mass Ratio 87
`Liquid Chromatography Resolves Proteins by Mass,
`Charge, or Binding Affinity 88
`Highly Specific Enzyme and Antibody Assays Can Detect
`Individual Proteins 90
`Radioisotopes Are Indispensabk Tools for Detecting
`Biological Molecules 90
`Protein Primary Structure Can Be Determined by
`Chemical Methods and from Gene Sequences 94
`Tnne-of-Flight Mass Spectrometry Measures the Mass of
`Proteins and Peptides 94
`Peptides with a Defined Sequence Can Be Synthesized
`Chemically 94
`/.
`Protein Conformatio~· Is Determined by Sophisticated
`r
`Physical Methods
`~~,
`
`1
`
`MEDIA CONNECTION S
`Focus: Chaperone-Mediated Folding
`Overview, Ufe Cyde of a Protein
`Technique: 505 6el Electrophoresis
`Technique: lmmunoblottlng
`Classical Experiment 3.1: Bringing an Enzymj! Back
`to Ute
`
`4 Nucleic Acids, the Genetic
`Code, and the Synthesis
`of Macromolecules
`4.1 Structure of Nucleic Acids 101
`Polymerization of Nucleotides Forms Nucleic Acids 101
`Native DNA ls a Double Helix of Complementary
`Antiparallel Chains 103
`DNA Can Undergo Reversible Strand Separation 105
`Many DNA Molecules Are Circular 107
`Local Unwinding of DNA Induces Supercoiling 108
`
`

`

`•
`
`RNA Molecules Exhibit.Varied Conformations and
`Functions 108
`
`4.2 Synthesis of Biopolymers: Rules of Macromolecular
`Carpentry 110
`
`3'
`
`4.3 Nucleic Acid Syqthesis 111
`Both DNA and RNA Chains Are Produced by Copying of
`Template DNA Strands 111
`Nucleic Acid Strands Grow in the 5' (cid:157)
`Direction 112
`RNA Polymerases Can Initiate Strand Growth but DNA
`Polymerases Cannot 112
`Replication of Duplex DNA Requires Assembly of Many
`Proteins at a Growing Fork 113
`Organization of Genes in DNA Differs in Proka.ryotes and
`Eukaryotes 114
`Eukaryotic Primary RNA Transcripts Ate Processed to
`Poem Functional mRNAs 115
`
`4.4 The Three Roles of RNA in Protein
`Synthesis 116
`Messenger RNA Carries Information from DNA in a
`Three-Letter Genetic Code 117
`Experiments with Synthetic mRNAs and Trinucleotides
`Broke the Genetic Code 119
`The Folded Structure of tRNA Promotes Its Decoding
`Functions 120
`Nonstandard Base l?aidng Often Occurs between Codons
`and Anticodons 122
`Aminoacyl-tRNA Synthetases Activate Amino Acids by
`Linking Them to tRNAs 123
`Each tRNA Molecule Is Recognized by a Specific
`Aminoacyl-tRNA Synthetase 124
`Ribosomes Are Protein-Synthesizing Machines 125
`
`4.5 Stepwise Formation of Proteins on
`Ribosomes 128
`The AUG Sta.rt Codon Is Recognized by Methionyl(cid:173)
`tRN~ec 128
`Bacterial Initiation of Protein Synthesis Begins Near a
`·
`Shine-Dalgamo Sequence in mRNA 129
`Eukaryotic Initiation of Protein Synthesis Occurs at the 5'
`End and Internal Sites in mRNA- 130
`During Chain Elongation Each Incoming Aminoacyl(cid:173)
`tRNA Moves th.rough Three Ribosomal Sites 131
`Protein Synthesis ls Terminated by Release Factors When
`a Stop Codon Is Reached 132
`Simultaneous Translation by Multiple Ribosomes and
`Their Rapid Recycling Increase the Efficiency of
`Protein Synthesis 133
`
`MEDIA CONNECTIONS
`Focus: Basic Transcriptional Mechanism
`Overview: Life Cyde of an mRNA
`Focus: Protein Synthesis
`Cla!!islc Experiment: 4.1: Cracking the Genet:lc Code
`
`Contents xix
`
`---------~-
`5 Biomembranes and the
`Subcellular Organization
`of Eukaryotic Cells
`
`5.1 Microscopy and Cell Architecture 140
`Light Microscopy Can Distinguish Objects Separated by
`0.2 µ,m or More 140
`Samples for Light Microscopy -OsuaUy Are Fixed,
`Sectiorled, and Stained 141
`Fluorescence Microscopy Can Localize ·and Quantify
`Specific Molecules in Cells 142
`Confocal Scanning and Deconvolution Microscopy
`Provide Sharper Images of Three-Dimensional
`Objects 144
`Phase-Contrast and Nomarski Interference Microscopy
`Visualize Unstained Living Cells 146
`Transmission Electron Microscopy Has a Limit of
`Resolution of 0,1 nm 147
`Scanning Electron Microscopy Visualizes Details on the
`Surfaces of CelJs and Particl~ 152
`
`5.2 Purification of Cells and Their Parts 152
`Flow Cytometry Separates Different Cell Types 153
`Disruption of Cells Releases Their Organelles and Other
`Contents 153
`Different Organelles Can B.e Separated by
`Centrifugation 154
`Organelle-Specific Antibodies Are Useful in Preparing
`Highly Purified Organelles 157
`
`5.3 Biomembranes: Structural Organization and Basic
`Functions 157
`.Phospholipids Are the MaIJ uipid Constituents of Most
`Biomembranes 157'1
`1
`Ev4ry Cellular Membrane Fofins a Closed Compartment
`I and Has a Cytosolic ~d an Exoplasmic Face 160
`Several :fypes of Evidence Point to the Universality of the
`Phospholipid Bilayer 160
`All Intl:gral Proteins and Gfyc;olipids Bind Asymmetrically
`to thr Lipid Bilayer 162
`The t'hospholipid Composition Differs in Two Membrane
`Leaflets 162
`Mos.t Lipids and Integral Proteins Are Laterally Mobile in
`8iomembranes 162
`Flui~ty of Membranes Depends on Temperature and
`Composition 164
`Me)illbrane Leaflets Can Be Separated and Each Face
`Viewed Individually 165
`The Plasma Membrane Has Many Common Functions in
`1All Cells 166
`
`5.4 O;ganelles of the Eukaryotic Cell 168
`Lysosomes Are Acidic Organelles That Contain a Battery
`of Degradative Enzymes 169
`
`

`

`XX Contents
`
`Plant Vacuoles Store Small Molecules an d Enable the CeU
`ro Elongate Rapidly 170
`Pcroxisomes Degrade Fatty Acids and Toxic
`Compounds 171
`Mitochondria Are the Principal Sites of ATP Production
`in Aerobic Cells 171
`Chloroplasts, the Sires of Photosynthesis, Contain Three
`Membrane-Limited Compartments 172
`The Endoplasmic Reticulum Is a Network of
`loterconnected Internal Membranes 172
`Golgi Vesicles Process and Sorr Secretory and Membrane
`Proteins 1 73
`T.he Double-Membraned Nucleus Contains the Nucleolus
`and a Fibrous Matrix 174
`The Cytosol Contains Many Particles and Cytoskeleral
`Fibers 175
`
`MEDIA CONNECTIONS
`Overview, Protein Secretion
`Technique: Reporter Constructs
`Classlc Experiment S.1: Separating Organelles
`
`(J Manipulating Cells and
`Viruses in Culture
`6.1 Growth of Microorganisms in Culture 181
`Many Microorganisms Can Be Grown in Minimal
`Medium 181
`Mutant Strains of Bacteria and Yeast Can Be Isolated by
`Replica Plating 182
`
`6.2 Growth of Animal Cells in Culture 183
`Rich Media Are Reg uired for Culture of Animal Cells 18 3
`Most Cultured Animal Cells Grow Only on Special Solid
`Sudaces 183
`Primary Cell Cultures Are Useful, but Have a Finite Life
`Span 185
`Transformed Cells Can Grow Indefinitely in
`Culture 186
`Fusion of Cultured Animal Cells Can Yield Ioterspecific
`Hybrids Useful in Somatic-Cell Genetics 187
`Hybrid Cells Often Are Selected in HAT Medium 189
`Hybridomas Are Used to Produce Monoclonal
`Antibodies 189
`
`6.3 Viruses: Structure, Function, and Uses 191
`Viral Capsids Are Regular Arrays of One or a Few Types
`of Protein 192
`Most Viral Host Ranges Are Narrow 194
`Viruses Can Be Cloned and Counted in Plaque
`Assays 194
`Vual Growth Cycles Are Classified as Lytic or
`Lysogenic. 194
`four Types of Bacterial Viruses /I.re Widely Used in
`Biochemical and Genetic Researd1 196
`
`Animal Viruses Are Classified by Genome Type and
`mRNA Synthesis Pathway 199
`Viral Vectors Can Be Used to Introduce Specific Genes
`into Cells 203
`
`MEDIA CDNNECTION5
`Technique: Preparing Monoclonal Ant.lbodle§
`Overview: Life Cycle of a Retrovirus
`Classic Experiment 6.1: The Discovery of Reverse
`Transcrlptase
`
`71 Recombinant DNA and Genomics
`
`7.1 DNA Cloning with Plasmid Vectors 208
`Plasmids Are Extrachromosomal Self-Replicating DNA
`Molecules 209
`E. Coli Plasmids Can Be Engineered for Use as Cloning
`Vectors 209
`Plasmid Cloning Permits Isolation of DNA Fragments
`from Complex Mixtures 210
`Restriction Enzymes Cut DNA Molecules at Specific
`Sequences 211
`•
`Restriction Fragments with Complementary "Sticky Ends"
`Ace Ligated Easily 212
`Polylinkers Facilitate Insertion of Restriction Fragments
`into Plasmid Vectors 214
`Small DNA Molecules Can Be Chemically
`Synthesized 215
`
`7.2 Constructing DNA Libraries with A Phage and
`Other Cloning Vectors 216
`Bacteriophage ,\ Can Be Modified foe Use as a Cloning
`Vector and Assembled in Vitro 216
`Nearly Complete Ge11o~ci Libraries of l-ligher Organisms
`Cao Be Prepared 0y ,\ Cloning 218
`cDNA Libraries Are P·cepa'red from Isolated
`'
`mRNAs 219
`·
`Larger DNA Fragments Can Be Cloned in Cosmids and
`Other Vectors 221
`
`7.3 Identifying, Analyzing, and Sequencing
`Cloned DNA 223
`Libraries Cao Be Screened with Membrane-Hybridization
`Assay 224
`Oligonucleotide Probes Ar.e Designed Based on Partial
`Protein Sequences 225
`Specific Clones Can Ile Identified Based on Properties of
`the Encoded Proteins 22 7
`Gel Electrophoresis Resolves DNA Fragments of Differeut
`Size 228
`Multiple Restriction Sites Can Be Mapped on a Cloned
`DNA Fragment 230
`Pulsed-Field Gel Electrophoresis Separates Large DNA
`Molecules 231
`Purified DNA Molecules Can Be Sequenced Rapidly by
`Two Methods 231
`
`

`

`7 .4 Bioinformatics 235
`Stored Sequences Suggest Functions of Newly Identified
`Genes and Proteins 235
`Comparative Anal}rsis of Genomes Reveals Mucb about
`an Organism's Biology 236
`Homologous Proteins [nvolved in Genetic Information
`Processing Are Widely Disttibuted 238
`Many Yeast Genes Function in Intracellular Protein
`Targeting and Secretion 239
`The C. elegans Genome Encodes Numerous Proteins
`Specific to Multicellular Organisms 239
`
`7.5 Analyzing Specific Nucleic Acids in Complex
`Mixtures 240
`Southem Blotting Detects Specific DNA Fragments 240
`Northern nlotting Detects Specific RNAs 241
`Specific RNAs Can ~e Quantitated and Mapped on DNA
`by Nuclease Protection 241
`Transcription Start Sites Can Be Mapped by S1 Protection
`and Primer Extension 243
`
`7.6 Producing High Levels of Protdns from Cloned
`cDNAs 244
`E. coli fu<;prcssion Systems Can Produce Full-Length
`Proteins 244
`Eukaryotic Expression Systems Can Produce Proteins with
`Post-Translational Modifications 245
`Cloned cDNAs Can Be Translated in Vitro to Yield
`Labeled Proteins 245
`
`7.7 Polymerase Chain Reaction: An Alternative
`to Cloning 246
`PCR Amplification of Mutant Alleles Permits Their
`Detection in Small Samples 246
`DNA Sequences Can Be Amplified foe Use in Cloning and
`as Probes 247
`
`7.8 DNA Microarrays: Analyzing Genome-Wide
`Expression 248
`MEDIA CONNECTIONS
`Technique: Pla51111d Clonlng
`Technique: 0ldeoxy 5equ en dng of DNA
`Technique: Po lymerase Chain Reaction
`Classic Experim ent 7 .1: Unleashing the Pow er of
`Exp on ential Growth: The Palyme.rase Ch a in
`Reactio n
`Oasslc Experiment 7.'i!.: 0emon st:rat.tng 5eq uence-
`5peclflc Cleavage b y a Restriction En zy me
`
`8 Genetic Analysis in Cell Biology
`
`8.1 Mutations: Types and Causes 255
`Mutations Are Recessive or Dominant 255
`Iabcritance Patterns of Recessive and Dominant
`Mutations Differ 256
`Mutations Involve Large or Small DNA Alterations 257
`
`Contents xxi
`
`Mutations Occur Spontaneously and Can Be
`Induced 257
`Some Human Diseases Are Caused by Spontaneous
`Mutations 258
`8.2 Isolation and Analysis of Mutants 261
`Temperature-Sensitive Screens Can Isolate Lethal
`Mutations in Haploids 261
`Recessive Lethal Mutations in Diploids Can Be Screened
`by Use of Visible Markers 263
`Complementation Analysis Determines If Different
`Mutations Are in the Same Gene 264
`Metabolic and Other Pathways Can Be Genetically
`Dissected 265
`Suppressor Mutations Cao Identify Genes Encoding
`loteracting Proteins 265
`8.3 Genetic Mapping of Mutations 266
`Segregation Patterns Indicate Whether Mutations Are on
`the Sarne or Different Chromosomes 267
`Chromosomal Mapping Locates Mutations on l'arricular
`Chromosomes 268
`Recombinational Analysis Can Map Genes Relatiye to
`Each Other on a Chromosome 269
`DNA Polymorphisms Are Used to Map Human
`Mutations 271
`Some Chromosomal Abnormalities Can Be Mapped by
`Banding Analysis 272
`8.4 Molecular Cloning of Genes Defined by
`Mutations 274
`Cloned DNA Segments Near a Gene of Interest Are
`Identified by Various Methods 274
`Chromosome Walking ls Used to Isolate a Limited Region
`of Contiguous DNA 275
`Physical Maps of Entire <;!1t¢0}osom,es Can Be
`Constructed by Screenlng XAC Clones for Sequence-
`Tagged Sites 276
`·
`.. •l•
`Physical and Genetic Map's Cao Be Correlated with the
`Aid of Known Markers 277
`Further Analysis Is Needed to L_ocate a Mutation-Defined
`Gene in Cloned DNA 278
`Protein Srrucrure Is Deduced from cDNA Sequence 279
`8.5 Gene Replacement and Transgenic Animals 281
`Specific Sites in Cloned Genes Can Be .(\leered in
`Vitro 281
`DNA Is Transferred into Eukaryotic Cells in Various
`Ways 282
`Normal Genes Can Be Replaced wirh_ M utant AlJeles io
`Yeast and Mice 282
`Foreign Genes Can Be Introduced into Plants and
`Animals 287
`MEDIA CONNECTIONS
`Technique: In Vitro M utagen es:ls of Cloned Genes:
`Technique: Creating a Traru;genlc M ouse
`Cl ass:lc Experiment B.I, Expr essing For eign Gen es
`In M ice
`
`

`

`x:xii Contents
`
`PART II: Nuclear Control of
`Cellular Activity
`
`9 Molecular Structure of Genes and
`Chromosomes
`
`9.1 Molecular Definition of a Gene 295
`Bacterial Operons Produce Polycistronic mRNAs 295
`Most Eukaryotic mRNAs Are Monocistronic and Contain
`Introns 295
`Simple and Complex Transcription Units Are Found in
`Eukaryotic Genomes 296
`
`9.2 Chromosomal Organization of Genes and
`Noncoding DNA 297
`Genomes of Higher Eukaryotes Contain Much
`Nonfunctional DNA 297
`Cellular DNA Content Does Not Correlate with
`Phylogeny 298
`Protein-Coding Genes May Be Solitary or Belong to a
`Gene ,Family 299
`Tandemly Repeated Genes Encode rRNAs, tRNAs, and
`Histones 300
`Reassociation Experiments Reveal Three Major Fractions
`of Eukaryotic DNA 301
`Simple-Sequence DNAs Are Concentrated in Specific
`Chromosomal Locations 301
`DNA Fingerprinting Depends on Differences in Length of
`Simple-Sequence DNAs 302
`
`9.3 Mobile DNA 303
`Movement of Mobile Elements Involves a DNA or RNA
`Intermediate 304·
`Mobile Elements Thar Move as DNA Are Present in
`Prokaryotes and Eukatyotes 304
`Viral Retrotransposons Contain LTRs and Behave Like
`Retroviruses in the Genome 307
`Nonviral Retrotransposons Lack LTRs and Move by an
`Unusual Mechanism 308
`Retrotransposed Copies of Cellular RNAs Occur in
`Eukaryotic Chromosomes 312
`Mobile DNA Elements Probably Had a Significant
`In£luence on Evolution 312
`
`9.4 Functional Rearrangements in Chromosomal
`DNA 314
`Inversion of a Transcription-Control Region Switches
`Salmonella Flagellar Antigens 314
`Antibody Genes Are Assem,bled by Rea,ra.ngements of
`Germ-Line DNA 315
`Generalized DNA Amplification Produces Polytene
`Ch.,omosomes 318
`
`9.5 Organizing Cellular DNA into Chromosomes 320
`Most Bacterial Chromosomes Are Circular with One
`Replication Origin 320
`Eukaryotic Nuclear DNA Associates with Histooe
`Protei.ns to Form Chromatin 321
`Chromatin Exists in Extended and Condensed
`Forms 321
`Acetylation of Histone N-Termini Reduces Chromatin
`Condensation 323
`Eukaryotic Chromosomes Contain One Linear DNA
`Molecule 324
`
`9 .6 Morphology and Functional Elements of Eukaryotic
`Chromosomes 324
`Chromosome Number, Size, and Shape at Metaphase Are
`Species Specific 325
`Nonhistone Proteins Provide a Structural Scaffold for
`Long Chromatin Loops 325
`Chromatin Contains Small Amounts of Other Proteins in
`Addition to Histones and Scaffold Proteins 327
`Stained Chromosomes Have Characteristic Banding
`Patterns 327
`Chromosome Painting Distinguishes Each Homologous
`Pair by Color 328
`Heterochromatin Consists of Chromosome Regions That
`Do Not Uncoil 329
`Three Functional Elements Are Required for Replication
`and Stable Inheritance of Chromosomes 329
`Yeast Artificial Chromosomes Can Be Used to Clone
`Megabase DNA Fragments 331
`
`/
`
`I,
`9.7 Organelle DNAs 331 - 1
`Mitochondria Contain Multiple mtDNA Molecules 332
`Genes in mtDNA Exli:ibit Cytoplasmic Inheritance and
`Encode rRNAs, tRNAs, and Some Mitochondrial
`Proteins 333
`The Size and Coding Capatlty of mtDNA Vaty
`Considerably in Different Organisms 334
`Products of Mitochondrial Genes Are Not
`Exported 335
`Mitochondrial Genetic Codes Differ from the Standard
`Nuclear Code 335
`Mutations in Mitochond.rial DNA Cause Several Genetic
`Diseases in Man 336
`Chloroplasts Contain Large Circular DNAs Encoding
`More Than a Hundred Proteins 336
`
`MEDIA CDNNECTJDN5
`Focus, Retrovlral Reverse Transcription
`Focus: Three-Dimensional Packing of Nudear
`Chromo.somes
`Classic Experiment 9.1: Two 6enes Become One:
`Somatic Rearrangement of lmmunaglobln
`6enes
`
`

`

`j() Regulation of Transcription
`Initiation
`10.1 Bacterial Gene Control: The Jacob-lvlonod
`Model 342
`Enzymes Encoded at the lac Operon Can Be Induced
`and Repressed 342
`Mutations in Lael Cause Constitutive Expression of lac
`Operon 343
`Isolation of Operator Constitutive and Promoter
`Mutants Support Jacob-Monod Model 343
`Regulation of lac Operon Depends on Cis-Acting DNA
`Sequences and Trans-Acting Proteins 344
`Biochemical Experiments Confirm That Induction of the
`lac. Operon Leads to Increased Synthesis of lac
`mRNA 344
`
`10.2 Bacterial Transcription Initiation 346
`Footprinting and Gel-Shift Assays Identify Protein-DNA
`Interactions 446
`The lac Control Region Contains Three Critical Cis(cid:173)
`Acting Sites 347
`RNA Polymerase Binds to Specific Promoter Sequences
`to Initiate Transcription 347
`Differences in E. coli Promoter Sequences Affect
`Frequency of Transcription Initiation 349
`Binding of lac Repressor to the lac Operator Blocks
`Transcription Initiation 349
`Most Bacterial Repressors Are Dimers Containing a
`Helices That Insert into Adjacent Major Grooves of
`Operator DNA 349
`Ligand-Induced Conformational Changes Alter Affinity
`of Many Repressors for DNA 352
`Positive Control of the lac Operon [s Exerted by cAMP(cid:173)
`CAP 352
`Cooperative Binding of cAMP-CAP and RNA
`Polymerase to lac· Control Region Activates
`Transcription 353
`Transcription Control at AU Bacterial Promoters Involves
`Similar but Distinct Mechanisms
`354
`Transcription from Some Promoters Is Initiated by
`Alternative Sigma (u) Factors 355
`Many Bacterial Responses Are Controlled by Two(cid:173)
`Component Regulatory Systems 356
`
`10.3 Eukaryotic Gene Control: Purposes and Gen,eral
`Principles 358
`Most Genes in Higher Eukaryotes Are Regulated by
`Controlling Their Transcription 358
`'.Regulatory Elements in Eu.karyotic DNA Often Are
`Many Kilobases from Start Sites 360
`Three Eukaryotic Polymerases Catalyze Formation of
`Different RNAs 361
`The Largest Subunit in RNA Polymerase Il Has an
`Essential Carboxyl-Terminal Repeat 362
`
`Contents xxiii
`
`RNA Polymerase II Initiates Transcription at DNA
`Sequences Corresponding to the 5' Cap of
`mRNAs 362
`
`10.4 Regulatory Sequences in Eukaryotic Protein-Coding
`Genes 365
`TATA Box, Initiators, and CpG Islands Function as
`Promoters in Eukaryotic DNA 365
`Promoter-Proximal Elemepts Help Regulate Eukaryotic
`Genes 366
`Transcription by RNA Polymerase Il Ofte,n Is Stimulated
`by Distant Enhancer Sites 368
`_
`Most Eukaryotic Genes Are Regulated by Multiple
`Transcription-Control Elements 369
`
`10.5 Eukaryotic Transcription Activators and
`Repressors 370
`Biochemical and Genetic Techniques Have Been Used to
`Identify Transcription Factors 370
`Transcription Activators Are Modular Proteins Composed
`of Distinct Functional Domains 372
`DNA-Binding Domains Can Be Classified into
`Numerous Structural Types 373
`Heterodimeric Transcription Factors Increase Gene(cid:173)
`Control Options 376
`Activation Domains Exhibit Considerable Structural
`Diversity 377
`Multiprotein Complexes Form on Enhancers 378
`Many Repressors Are the Functional Converse of
`Activators 379
`
`10.6 RNA Polymerase II Transcription-Initiation
`Complex 380
`Initiation by Pol TT Requires General Transcription
`,. j ,
`Factors 381
`Proteins Comprising th_e 'f>ol,ll Transcription-Initiation
`Complex Assemble in a •Specific Order in Vitro 381
`A Pol II Holoenzyme M~ltip.totein Complex Functions in
`Vivo 383
`
`10.7 Molecular Mechanisms of Eukaryotic
`Transcriptional Control 384
`N-Termini of Histones in Chromatin Can Be
`Modified 384
`Formation of Heterochromatin Silences Gene Expression
`at Telomeres and Other Regions 384
`Repressors Can Direct Histone Deacecylatioa, at Specific
`Genes 387
`Activators Can Direct Histone Acetylation at Specific
`Genes 389
`Chromatin-Remodeling Factors Participate in Activatiop
`at Some Promoters 390
`Activators Stimulate the Highly Cooperative Assembly of
`Initiation Complexes 390
`Rep(essors Interfere Directly with Transcription
`Initiation in Several Ways 391
`
`

`

`XXIV Contents
`
`Regulation of Transcription-Factor Expression
`Contributes to Gene Control 392
`Lipid-Soluble Hormones Control the Activities of
`Nuclear Receptors 392
`Polypeptide Hormones Signal Phosphorylation of Some·
`Transcription Factors 394
`
`10.8 Other Transcription Systems 397
`Transcription Initiation by Pol I aod Pol ill Is Analogous
`to That by Pol IT 397
`T7 and Related Bacteriophages Express Monomeric,
`Largely Unregulated RNA Polymerases 398
`Mitochondrial DNA ls Transcribed by RNA Polymerases
`with Similarities to Bacteriophage and Bacterial
`Enzymes 398
`Transcription of Chloroplast DNA Resembles Bacterial
`Transcription 399
`Transcription by Archaeans Is Closer to Eukaryotic Than
`to Bacterial Transcription 399
`
`MEDIA CONNECTIONS
`FocU!i, Comblnatorlal Control of Transcription
`
`11 RNA Processing,
`Nuclear Transport, and Post(cid:173)
`Transcriptional Control
`11.1 Transcription Tecm.ination 405
`Rho-Independent Termination Occurs at Characteristic
`Sequences in E. coli DNA 405
`Premature Termination by Attenuation Helps Regulate
`Expression -0£ Some Bacterial Operon,s 405
`Rho-Dependent Termination Sites Are Present in Some
`A-Phage and E. coli Genes 407
`Sequence-Specific RNA-Binding Proteins Can Regulate
`Termination by E. coli RNA Poly,merase 407
`Three Eukaryotic RNA Polymer,ases Emp1oy Different
`Termination Mechanismr· 408
`Transcription of HlV Genome Is Regulated by an
`Antitermination Mechanism 409
`Promoter-Proximal Pausing of RNA .Pormerase ll
`Occurs in Some Rapidly Induced Genes 409
`11.2 Processing of Eukaryotic. mRNA 410
`The 5 1 -Cap Is Added to Nascent RNAs Shortly after
`Initiation by RNA Polymerase II 410
`Pre-mRNAs Are Associated with hnRNP Prate.ins
`Containing Conserved RNA-Binding Domains 410
`hnRNP Proteins May Assist in Processing and Transport
`ofmRNAs 413
`Pre-mRNAs Are Cleaved at Specific 3' Sites and Rapidly
`Polyadenylated 413
`Splicing Occurs at Short, Conserved Sequences in Pre(cid:173)
`mRNAs via Two Transesterlfication Reactions 415
`Spliceosomes, Assembled from snRNPs and a Pre(cid:173)
`mRNA, Carry Out Splicing 416
`
`Portions of Two Different RNAs Are Trans-Spliced in
`Some Organisms 418
`Self-Splicing Group Il lntrons Provide Clues to the
`Evolution of snRNAs 419
`Most Transcription and RNA Processing Occur in a
`Limited N umber of Dom.B,ins in Mammalian Cell
`Nuclei 420
`
`11.3 Regulation of mRNA Processing 422
`UlA Protein Inhibits Polyadenylation of Its Pre(cid:173)
`mRNA 422
`Tissue-Specific RNA Splicing Controls Expression of
`Alternative Fibronectins 423
`A Cascade of Regulated RNA Splicing Controls
`Drosophila Sexual Differentiation 423
`Multiple Protein Isoforms Are Common in the
`Vertebrate Nervous System 425
`
`11.4 Signal-Mediated Transport through N uclear Pore
`Complexes 426
`Nuclear Pore Complexes Actively Transport
`Macromolecules between the Nucleus and
`Cytoplasm 427
`•
`Receptors for Nuclear-Export Signals Transport Proteins
`and mRNPs out of the Nucleus 428
`Pre-mRNAs in Spliceosomes Are Not Exported from the
`Nucleus 431
`Receptors for Nuclear-Localization Signals Transport
`Proteins into the Nucleus 432
`Various Nuclear-Transport Systems Utilize Similar
`,Proreins 434
`HIV Rev Protein Reg