`
`FOURTH EDITION
`
`
`
`MOLECULAR
`CELL
`BIOLOGY
`
`
`
`
`
`
`
`sil
`
`Arnold Berk
`
`S. Lawrence Zipursky
`
`Paul Matsudaira
`
`S David Baltimore
`
`
`James Darnell
`
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`Paul Matsudaira
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`Arnold Berk
`S. Lawrence Zipursky.
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`ae” by W. H. Freeman
`Toe TaeREE
`— acne
`Alnylam Exh. 1059
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`Ra W. H. FREEMAN AND COMPA
`
`I
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`Alnylam Exh. 1059
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`Executive Epiror: Sara Tenney
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`
`Library of Congress Cataloging-in-Publication Data
`
`Molecularcell 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-de21
`
`I. Lodish, Harvey F
`
`99-30831
`CIP
`
`© 1986, 1990, 1995, 2000 by W. H. Freeman and Company.All rights reserved.
`
`Nopart of this book may be reproduced by any mechanical, photographic, or
`electronic process, or in the form of a phonographic recording, nor mayit 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
`
`Secondprinting, 2000
`
`

`

`,ontents in
`
`rie
`
`PART | Laying the Groundwork
`
`PART II! Building and Fueling
`the Cell
`
`15
`
`16
`
`17
`
`18
`
`19
`
`Transport across Cell Membranes
`
`578
`
`Cellular Energetics: Glycolysis, Aerobic
`Oxidation, and Photosynthesis
`616
`
`Protein Sorting: Organelle Biogenesis and
`Protein Secretion 675
`
`Gell Motility and Shape I:
`Microfilaments
`751
`
`Cell Motility and Shape II: Microtubules
`and Intermediate Filaments
`795
`
`PART IV Cell Interactions
`
`20
`
`21
`
`22
`
`23
`
`24
`
`Cell-to-Cell Signaling: Hormones and
`Receptors
`848
`
`Nerve Cells
`
`911
`
`Integrating Cells into Tissues
`
`968
`
`Cell Interactions in Development
`
`1003
`
`Cancer
`
`1054
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`The Dynamic Cell
`
`1
`
`Chemical Foundations
`
`14
`
`Protein Structure and Function 50
`
`Nucleic Acids, the Genetic Code, and the
`Synthesis of Macromolecules
`100
`
`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
`
`PART Il Nuclear Control of
`Cellular Activity
`
`9
`
`10
`
`11.
`
`12
`
`Molecular Structure of Genes and
`Chromosomes
`294
`
`Regulation of Transcription Initiation 341
`
`RNA Processing, Nuclear Transport, and
`Post-Transcriptional Control
`404
`
`DNAReplication, Repair, and
`Recombination 453
`
`13
`
`Regulation of the Eukaryotic Cell Cycle
`
`495
`
`14 Gene Control in Development
`
`537
`
`I,
`
`;
`
`}
`
`i
`'
`
`'
`;
`
`i
`
`

`

`Contents
`
`Chapter-OpeningIllustrations XXXVI
`
`PART |: Laying the Groundwork
`
`2.2
`
`1
`
`The Dynamic Cell
`
`fal
`
`Evolution: At the Core of Molecular Change
`
`3
`
`1.2
`
`The 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
`Prokaryotes Comprise a Single Membrane-Limited
`Compartment
`7
`Eukaryotic Cells Contain Many Organelles and a
`Complex Cytoskeleton
`7
`Cellular DNA Is Packaged within Chromosomes
`
`8
`
`6
`
`9
`The Life Cycle of Cells
`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
`Cells Die by Suicide
`10
`
`10
`
`Cells into Tissues
`11
`Multicellularity Requires Extracellular Glues
`Tissues Are Organized into Organs
`11
`Body Plan and Rudimentary Tissues Form Early in
`Embryonic Development
`12
`
`11
`
`1.4
`
`1.5
`
`1.6
`
`Molecular Cell Biology: An Integrated View of Cells
`at Work
`13
`
`MEDIA CONNECTIONS
`
`Overview: Life Cycle of a Cell
`
`2
`
`Chemical Foundations
`
`2.1
`
`15
`Covalent Bonds
`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
`Electrons Are Shared Unequally in Polar Covalent
`Bonds
`18
`
`17
`
`Asymmetric Carbon Atoms Are Present in Most Biological
`Molecules
`19
`a and B Glycosidic Bonds Link Monosaccharides
`
`21
`
`Noncovalent 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 Can Confer Binding
`Specificity
`26
`26
`Phospholipids Are Amphipathic Molecules
`The Phospholipid Bilayer Forms the Basic Structure of All
`Biomembranes
`27
`
`2.3
`
`Chemical Equilibrium 29
`Equilibrium 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-Hasselbalch Equation Relates pH and Keg
`of an Acid-Base System 33
`Buffers Maintain the pH of Intracellular and Extracellular
`Fluids
`33
`
`2.4
`
`35
`Biochemical Energetics
`Living Systems Use Various Forms of Energy, Which Are
`Interconvertible
`35
`
`The Change in Free Energy AG Determines the Direction
`of a Chemical Reaction
`36
`The AG of a Reaction Depends on Changes in Enthalpy
`(Bond Energy) and Entropy
`36
`Several Parameters Affect the AG of a Reaction
`37
`The AG*of a Reaction Can Be Calculated from Its K.g
`Cells Must Expend Energy to Generate Concentration
`Gradients
`39
`
`38
`
`ManyCellular Processes Involve Oxidation-Reduction
`Reactions
`39
`
`An Unfavorable Chemical Reaction Can ProceedIf It Is
`Coupled with an Energetically Favorable Reaction
`Hydrolysis of Phosphoanhydride Bonds in ATP Releases
`Substantial Free Energy
`41
`ATP Is Used to Fuel Many Cellular Processes
`
`43
`
`41
`
`cena
`
`

`

`Xvlii|Contents
`
`45
`2.5 Activation Energy and Reaction Rate
`Chemical Reactions Proceed through High-Energy
`Transition States
`45
`Enzymes Accelerate Biochemical Reactions by Reducing
`Transition-State Free Energy
`47
`
`MEDIA CONNECTIONS
`Overview: Biological Energy Interconversions
`
`3 Protein Structure and Function
`
`51
`3.1 Hierarchical Structure of Proteins
`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 Representationsof 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 Folding Is Encodedin the
`Sequence
`63
`Folding of Proteins in Vivo Is Promoted by
`Chaperones
`63
`Chemical Modifications and Processing Alter the
`Biological Activity of Proteins
`64
`66
`Cells Degrade Proteins via Several Pathways
`Aberrantly Folded Proteins Are Implicated in Slowly
`Developing Diseases
`67
`68
`3.3 Functional Design of Proteins
`Proteins Are Designed to Bind a Wide Range of
`Molecules
`68
`Antibodies Exhibit Precise Ligand-Binding
`Specificity
`70
`71
`Enzymes Are Highly Efficient and Specific Catalysts
`An Enzyme’s Active Site Binds Substrates and Carries Out
`Catalysis
`71
`Kinetics of an Enzymatic Reaction Are Described by Vinax
`and K,,
`73
`ManyProteins Contain Tightly Bound Prosthetic
`Groups
`74
`A Variety of Regulatory Mechanisms Control Protein
`Function
`75
`
`
`
`By
`
`78
`3.4 Membrane Proteins
`78
`Proteins Interact with Membranes in Different Ways
`Hydrophobic @ Helices in TransmembraneProteins Are
`Embedded in the Bilayer
`79
`ManyIntegral Proteins Contain Multiple Transmembrane
`a Helices
`79
`Multiple 8 Strands in Porins Form Membrane-Spanning
`“Barrels”
`81
`Covalently Attached Hydrocarbon Chains Anchor Some
`Proteins to the Membrane
`81
`Some Peripheral Proteins Are Soluble Enzymes That Act
`on Membrane Components
`82
`Purifying, Detecting, and Characterizing
`Proteins
`83
`Proteins Can Be Removed from Membranes by Detergents
`or High-Salt Solutions
`83
`Centrifugation Can Separate Particles 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 Indispensable Tools for Detecting
`Biological Molecules
`90
`Protein Primary Structure Can Be Determined by
`94
`Chemical Methods and from Gene Sequences
`Time-of-Flight Mass Spectrometry Measures the Mass of
`Proteins and Peptides
`94
`Peptides with a Defined Sequence Can Be Synthesized
`Chemically
`94
`Protein Conformation Is Determined by Sophisticated
`Physical Methods
`95
`MEDIA CONNECTIONS
`Focus: Chaperone-Mediated Folding
`Overview: Life Cycle of a Protein
`Technique: SDS Gel Electrophoresis
`Technique: Immunoblotting
`Classical Experiment 3.1: Bringing an Enzyme Back
`to Life
`
`4.1
`
`Nucleic Acids, the Genetic
`Code, and the Synthesis
`of Macromolecules
`101
`Structure of Nucleic Acids
`Polymerization of Nucleotides Forms Nucleic Acids
`Native DNA Is a Double Helix of Complementary
`Antiparallel Chains
`103
`DNA Can Undergo Reversible Strand Separation
`Many DNA Molecules Are Circular
`107
`Local Unwinding of DNA Induces Supercoiling
`
`101
`
`105
`
`108
`
`

`

`Contents|xix
`
`5
`
`5.1
`
`Biomembranes and the
`Subcellular Organization
`of Eukaryotic Cells
`
`140
`Microscopy and Cell Architecture
`Light Microscopy Can Distinguish Objects Separated by
`0.2 wm or More
`140
`Samples for Light Microscopy Usually Are Fixed,
`Sectioned, 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 Cells and Particles
`152
`
`Sea
`
`152
`Purification of Cells and Their Parts
`153
`Flow Cytometry Separates Different Cell Types
`Disruption of Cells Releases Their Organelles and Other
`Contents
`153
`
`Different Organelles Can Be Separated by
`Centrifugation
`154
`Organelle-Specific Antibodies Are Useful in Preparing
`Highly Purified Organelles
`157
`
`RNA Molecules Exhibit Varied Conformations and
`Functions
`108
`
`4.2
`
`Synthesis of Biopolymers: Rules of Macromolecular
`Carpentry
`110
`
`111
`4,3 Nucleic Acid Synthesis
`Both DNA and RNA Chains Are Produced by Copying of
`Template DNA Strands
`111
`Nucleic Acid Strands Grow in the 5’ > 3’
`Direction
`112
`
`RNAPolymerases 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 Prokaryotes and
`Eukaryotes
`114
`Eukaryotic Primary RNA Transcripts Are Processed to
`Form 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 mRNAsand Trinucleotides
`Broke the Genetic Code
`119
`The Folded Structure of tRNA PromotesIts Decoding
`Functions
`120
`
`Nonstandard Base Pairing 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 Specifi
`Aminoacyl-tRNA Synthetase
`124
`Ribosomes Are Protein-Synthesizing Machines
`
`125
`
`4.5 Stepwise Formation of Proteins on
`Ribosomes
`128
`The AUGStart Codon Is Recognized by Methionyl-
`tRNAM*
`128
`Bacterial Initiation of Protein Synthesis Begins Near a
`Shine-Dalgarno 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-
`tRNA Moves through Three Ribosomal Sites
`131
`Protein Synthesis Is 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
`
`so
`
`Biomembranes: Structural Organization and Basic
`Functions
`157
`Phospholipids Are the Main Lipid Constituents of Most
`Biomembranes
`157
`Every Cellular Membrane Forms a Closed Compartment
`and Has a Cytosolic and an Exoplasmic Face
`160
`Several ‘Types of Evidence Point to the Universality of the
`Phospholipid Bilayer
`160
`All Integral Proteins and Glycolipids Bind Asymmetrically
`to the Lipid Bilayer
`162
`The Phospholipid Composition Differs in Two Membrane
`Leaflets
`162
`Most Lipids and Integral Proteins Are Laterally Mobile in
`Biomembranes
`162
`Fluidity of Membranes Depends on Temperature and
`Composition
`164
`MembraneLeaflets Can Be Separated and Each Face
`Viewed Individually
`165
`The Plasma Membrane Has Many Common Functions in
`jAll Cells
`166
`168
`Organelles of the Eukaryotic Cell
`Lysosomes Are Acidic Organelles That Contain a Battery
`of Degradative Enzymes
`169
`
`Focus: Basic Transcriptional Mechanism
`Overview: Life Cycle of an mRNA
`Focus: Protein Synthesis
`- Classic Experiment 4.1: Cracking the Genetic Code
`
`5.4
`
`

`

`XX Contents
`
`Plant Vacuoles Store Small Molecules and Enable the Cell
`to Elongate Rapidly
`170
`Peroxisomes Degrade Fatty Acids and Toxic
`Compounds
`171
`Mitochondria Are the Principal Sites of ATP Production
`in Aerobic Cells
`171
`Chloroplasts, the Sites of Photosynthesis, Contain Three
`Membrane-Limited Compartments
`172
`The Endoplasmic Reticulum Is a Network of
`Interconnected Internal Membranes
`172
`Golgi Vesicles Process and Sort Secretory and Membrane
`Proteins
`173
`The Double-Membraned Nucleus Contains the Nucleolus
`and a Fibrous Matrix
`174
`The Cytosol Contains ManyParticles and Cytoskeletal
`Fibers
`175
`
`MEDIA CONNECTIONS
`Overview: Protein Secretion
`
`Technique: Reporter Constructs
`Classic Experiment 5.1: Separating Organelles
`
`Manipulating Cells and
`Viruses in Culture
`
`181
`6.1 Growth of Microorganisms in Culture
`Many Microorganisms Can Be Grown in Minimal
`Medium 181
`Mutant Strains of Bacteria and Yeast Can Be Isolated by
`Replica Plating
`182
`
`183
`6.2 Growth of Animal Cells in Culture
`183
`Rich Media Are Required for Culture of Animal Cells
`Most Cultured Animal Cells Grow Only on Special Solid
`Surfaces
`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 Interspecific
`Hybrids Useful in Somatic-Cell Genetics
`187
`Hybrid Cells Often Are Selected in HAT Medium 189
`Hybridomas Are Used to Produce Monoclonal
`Antibodies
`189
`
`Gd
`
`191
`Viruses: Structure, Function, and Uses
`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
`Viral Growth Cycles Are Classified as Lytic or
`_ Lysogenic
`194
`Four Types of Bacterial Viruses Are Widely Used in
`Biochemical and Genetic Research
`196
`
`Animal Viruses Are Classified by Genome Type and
`mRNASynthesis Pathway
`199
`Viral Vectors Can Be Used to Introduce Specific Genes
`into Cells
`203
`
`MEDIA CONNECTIONS
`
`Technique: Preparing Monoclonal Antibodies
`Overview: Life Cycle of a Retrovirus
`Classic Experiment 6.1: The Discovery of Reverse
`Transcriptase
`
`yh
`
`Recombinant DNA and Genomics
`
`Fall
`
`Take
`
`Ted
`
`208
`DNA Cloning with Plasmid Vectors
`Plasmids Are ExtrachromosomalSelf-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”
`Are Ligated Easily
`212
`Polylinkers Facilitate Insertion of Restriction Fragments
`into Plasmid Vectors
`214
`Small DNA Molecules Can Be Chemically
`Synthesized
`215
`Constructing DNA Libraries with A Phage and
`Other Cloning Vectors
`216
`Bacteriophage A Can Be Modified for Use as a Cloning
`Vector and Assembled in Vitro
`216
`Nearly Complete Genomic Libraries of Higher Organisms
`Can Be Prepared by A Cloning
`218
`cDNALibraries Are Prepared from Isolated
`mRNAs
`219
`Larger DNA Fragments Can Be Cloned in Cosmids and
`Other Vectors
`221
`
`Identifying, Analyzing, and Sequencing
`Cloned DNA 223
`Libraries Can Be Screened with Membrane-Hybridization
`Assay
`224
`Oligonucleotide Probes Are Designed Based on Partial
`Protein Sequences
`225
`Specific Clones Can Be Identified Based on Properties of
`the Encoded Proteins
`227
`Gel Electrophoresis Resolves DNA Fragments ofDifferent
`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
`
`

`

`
`
`Contents | xxi
`
`235
`7.4 Bioinformatics
`Stored Sequences Suggest Functions of Newly Identified
`Genes and Proteins
`235
`Comparative Analysis of Genomes Reveals Much about
`an Organism’s Biology
`236
`Homologous Proteins Involved in Genetic Information
`Processing Are Widely Distributed
`238
`Many Yeast Genes Function in Intracellular Protein
`Targeting and Secretion
`239
`The C. elegans Genome Encodes NumerousProteins
`Specific to Multicellular Organisms
`239
`
`7.5 Analyzing Specific Nucleic Acids in Complex
`Mixtures
`240
`Southern Blotting Detects Specific DNA Fragments
`Northern Blotting Detects Specific RNAs
`241
`Specific RNAs Can Be Quantitated and Mapped on DNA
`by Nuclease Protection
`241
`Transcription Start Sites Can Be Mapped by S1 Protection
`and Primer Extension
`243
`
`240
`
`7.6 Producing High Levels of Proteins from Cloned
`cDNAs
`244
`E. coli Expression 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 Smail Samples
`246
`DNA Sequences Can Be Amplified for Use in Cloning and
`as Probes
`247
`
`7.8 DNA Microarrays: Analyzing Genome-Wide
`Expression
`248
`MEDIA CONNECTIONS
`
`Technique: Plasmid Cloning
`Technique: Dideoxy Sequencing of DNA
`Technique: Polymerase Chain Reaction
`Classic Experiment 7.1: Unleashing the Power of
`Exponential Growth: The Polymerase Chain
`Reaction
`
`Classic Experiment 7.2: Demonstrating Sequence-
`Specific Cleavage by a Restriction Enzyme
`
`|
`
`Genetic Analysis in Cell Biology
`
`255
`8.1 Mutations: Types and Causes
`Mutations Are Recessive or Dominant
`
`255
`
`Inheritance Patterns of Recessive and Dominant
`Mutations Differ
`256
`
`Mutations Involve Large or Small DNA Alterations
`
`257
`
`Mutations Occur Spontaneously and Can Be
`Induced
`257
`
`Some Human Diseases Are Caused by Spontaneous
`Mutations
`258
`
`8.2
`
`261
`Isolation and Analysis of Mutants
`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
`
`8.3
`
`Metabolic and Other Pathways Can Be Genetically
`Dissected
`265
`
`Suppressor Mutations Can Identify Genes Encoding
`Interacting Proteins
`265
`266
`Genetic Mapping of Mutations
`Segregation Patterns Indicate Whether Mutations Are on
`the Same or Different Chromosomes
`267
`Chromosomal Mapping Locates Mutations on Particular
`Chromosomes
`268
`
`Recombinational Analysis Can Map GenesRelative 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
`Molecular Cloning of Genes Defined by
`Mutations
`274
`
`8.4
`
`Cloned DNA Segments Near a Geneof Interest Are
`Identified by Various Methods
`274
`Chromosome Walking Is Used to Isolate a Limited Region
`of Contiguous DNA 275
`Physical Maps of Entire Chromosomes Can Be
`Constructed by Screening YAC Clones for Sequence-
`Tagged Sites
`276
`Physical and Genetic Maps Can Be Correlated with the
`Aid of Known Markers
`277
`
`Further Analysis Is Needed to Locate a Mutation-Defined
`Gene in Cloned DNA 278
`
`8.5
`
`279
`Protein Structure Is Deduced from cDNA Sequence
`Gene Replacement and Transgenic Animals
`281
`Specific Sites in Cloned Genes Can Be Altered in
`Vitro
`281
`
`DNAIs Transferred into Eukaryotic Cells in Various
`Ways
`282
`Normal Genes Can Be Replaced with Mutant Alleles in
`Yeast and Mice
`282
`
`Foreign Genes Can Be Introduced into Plants and
`Animals
`287
`MEDIA CONNECTIONS
`
`Technique: In Vitro Mutagenesis of Cloned Genes
`Technique: Creating a Transgenic Mouse
`Classic Experiment 8.1: Expressing Foreign Genes
`in Mice
`
`
`
`BB
`
`

`

`xxii
`
`| Contents
`
`PART II: Nuclear Control of
`Cellular Activity
`
`9
`
`a.
`
`DL
`
`Molecular Structure of Genes and
`Chromosomes
`
`295
`Molecular Definition of a Gene
`295
`Bacterial Operons Produce Polycistronic mRNAs
`Most Eukaryotic mRNAs Are Monocistronic and Contain
`Introns
`295
`Simple and Complex Transcription Units Are Found in
`Eukaryotic Genomes
`296
`
`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 MayBeSolitary 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
`DNAFingerprinting Depends on Differences in Length of
`Simple-Sequence DNAs
`302
`
`33
`
`Mobile DNA 303
`Movement of Mobile Elements Involves a DNA or RNA
`Intermediate
`304
`Mobile Elements That Move as DNA Are Present in
`Prokaryotes and Eukaryotes
`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
`Influence 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 Assembled by Rearrangements of
`Germ-Line DNA 315
`Generalized DNA Amplification Produces Polytene
`Chromosomes
`318
`
`9.5
`
`9.6
`
`320
`
`Organizing Cellular DNA into Chromosomes
`Most Bacterial Chromosomes Are Circular with One
`Replication Origin
`320
`Eukaryotic Nuclear DNA Associates with Histone
`Proteins 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
`
`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
`ChromosomePainting 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
`
`9.7
`
`332
`Organelle DNAs
`332
`Mitochondria Contain Multiple mtDNA Molecules
`Genes in mtDNA Exhibit Cytoplasmic Inheritance and
`Encode rRNAs, tRNAs, and Some Mitochondrial
`Proteins
`333
`The Size and Coding Capacity of mtDNA Vary
`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 Mitochondrial DNA Cause Several Genetic
`Diseases in Man
`336
`Chloroplasts Contain Large Circular DNAs Encoding
`More Than a Hundred Proteins
`336
`
`MEDIA CONNECTIONS
`Focus: Retroviral Reverse Transcription
`Focus: Three-Dimensional Packing of Nuclear
`Chromosomes
`
`Classic Experiment 9.1: Two Genes Become One:
`Somatic Rearrangement of Immunogloabin
`Genes
`
`Ce
`
`

`

`Contents|xxiii
`
`10 Regulation of Transcription
`Initiation
`
`10.1
`
`Bacterial Gene Control: The Jacob-Monod
`Model
`342
`Enzymes Encoded at the Jac Operon Can Be Induced
`and Repressed
`342
`Mutations in /acI 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
`
`346
`Bacterial Transcription Initiation
`Footprinting and Gel-Shift Assays Identify Protein-DNA
`Interactions
`346
`
`The lac Control Region Contains Three Critical Cis-
`Acting Sites
`347
`RNAPolymerase 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 Jac Operator Blocks
`Transcription Initiation
`349
`Most Bacterial Repressors Are Dimers Containing oa
`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 Is Exerted by cAMP-
`CAP
`352
`
`Cooperative Binding of cAMP-CAP and RNA
`Polymerase to lac Control Region Activates
`Transcription
`353
`Transcription Control at All Bacterial Promoters Involves
`Similar but Distinct Mechanisms
`354
`Transcription from Some Promoters Is Initiated by
`Alternative Sigma (7) Factors
`355
`ManyBacterial Responses Are Controlled by Two-
`Component Regulatory Systems
`356
`
`10.3
`
`Eukaryotic Gene Control: Purposes and General
`Principles
`358
`Most Genes in Higher Eukaryotes Are Regulated by
`Controlling Their Transcription
`358
`Regulatory Elements in Eukaryotic DNA Often Are
`ManyKilobases from Start Sites
`360
`Three Eukaryotic Polymerases Catalyze Formation of
`Different RNAs
`361
`The Largest Subunit in RNA Polymerase II Has an
`362
`Essential Carboxyl-Terminal Repeat
`
`RNAPolymeraseII Initiates Transcription at DNA
`Sequences Corresponding to the 5’ Cap of
`mRNAs
`362
`
`10.4
`
`Regulatory Sequences in Eukaryotic Protein-Coding
`Genes
`365
`
`10.5
`
`TATA Box,Initiators, and CpG Islands Function as
`Promoters in Eukaryotic DNA 365
`Promoter-Proximal Elements Help Regulate Eukaryotic
`Genes
`366
`
`Transcription by RNA Polymerase II Often Is Stimulated
`by Distant Enhancer Sites
`368
`Most Eukaryotic Genes Are Regulated by Multiple
`Transcription-Control Elements
`369
`
`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
`NumerousStructural Types
`373
`Heterodimeric Transcription Factors Increase Gene-
`Control Options
`376
`Activation Domains Exhibit Considerable Structural
`Diversity
`377
`378
`Multiprotein Complexes Form on Enhancers
`Many Repressors Are the Functional Converse of
`Activators
`379
`
`10.6
`
`RNAPolymerase II Transcription-Initiation
`Complex
`380
`Initiation by Pol II Requires General Transcription
`Factors
`381
`
`10.7
`
`Proteins Comprising the Pol II Transcription-Initiation
`Complex Assemble in a Specific Order in Vitro
`381
`A Pol II Holoenzyme Multiprotein Complex Functions in
`Vivo
`383
`
`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 Deacetylation at Specific
`Genes
`387
`
`Activators Can Direct Histone Acetylation at Specific
`Genes
`389
`
`Chromatin-Remodeling Factors Participate in Activation
`at Some Promoters
`390
`Activators Stimulate the Highly Cooperative Assembly of
`Initiation Complexes
`390
`Repressors 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
`
`397
`Other Transcription Systems
`Transcription Initiation by Pol I and Pol IJ Is Analogous
`to That by Pol II
`397
`T7 and Related Bacteriophages Express Monomeric,
`Largely Unregulated RNA Polymerases
`398
`Mitochondrial DNA Is 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
`
`Focus: Combinatorial Control of Transcription
`
`1
`
`11.1
`
`11.2
`
`_ RNAProcessing,
`Nuclear Transport, and Post-
`Transcriptional Control
`
`405
`Transcription Termination
`Rho-Independent Termination Occurs at Characteristic
`Sequences in E. coli DNA 405
`Premature Termination by Attenuation Helps Regulate
`Expression of Some Bacterial Operons
`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 Polymerase
`407
`Three Eukaryotic RNA Polymerases Employ Different
`Termination Mechanisms+ 408
`Transcription of HIV GenomeIs Regulated by an
`Antitermination Mechanism 409
`Promoter-Proximal Pausing of RNA Polymerase II
`Occurs in Some Rapidly Induced Genes
`409
`
`Processing of Eukaryotic mRNA 410
`The 5’-Cap Is Added to Nascent RNAsShortly after
`Initiation by RNA Polymerase II
`410
`Pre-mRNAs Are Associated with hnRNP Proteins
`410
`Containing Conserved RNA-Binding Domains
`hnRNP Proteins MayAssist in Processing and Transport
`of mRNAs
`413
`Pre-mRNAsAre Cleaved at Specific 3’ Sites and Rapidly
`Polyadenylated
`413
`Splicing Occurs at Short, Conserved Sequences in Pre-
`mRNAsvia Two Transesterification Reactions
`415
`
`Spliceosomes, Assembled from snRNPs and a Pre-
`mRNA,Carry Out Splicing
`416
`
`Portions of Two Different RNAs Are Trans-Spliced in
`Some Organisms
`418
`Self-Splicing Group II Introns Provide Clues to the
`Evolution of snRNAs
`419
`
`Most Transcription and RNA Processing Occur in a
`Limited Number of Domains in Mammalian Cell
`Nuclei
`420
`
`11.3
`
`422
`Regulation of mRNA Processing
`U1AProtein Inhibits Polyadenylation of Its Pre-
`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 Commonin the
`Vertebrate Nervous System 425
`
`11.4
`
`11.5
`
`Signal-Mediated Transport through Nuclear Pore
`Complexes
`426
`Nuclear Pore Complexes Actively Transport
`Macromolecules between the Nucleus and
`Cytoplasm 427
`Receptors for Nuclear-Export Signals Transport Proteins
`and mRNPsoutof the Nucleus
`428
`Pre-mRNAsin 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
`Proteins
`434
`
`HIV Rev Protein Regulates the Transport of Unspliced
`Viral mRNAs
`435
`
`437
`
`Other Mechanisms of Post-Transcriptional
`Control
`436
`RNA Editing Alters the Sequences of PreemRNAs
`Some mRNAsAre Associated with Cytoplasmic
`Structures or Localized to Specific Regions
`Stability of Cytoplasmic mRNAs Varies Widely
`Degradation Rate of Some Eukaryotic mRNAsIs
`Regulated
`440
`Translation of Some mRNAsIs Regulated by Specific
`RNA-Binding Proteins
`442
`Antisense RNA Regulates Translation of Transposase
`mRNAin Bacteria
`442
`
`438
`440
`
`11.6
`
`Processing of rRNA and tRNA 443
`Pre-rRNA Genes Are Similar in All Eukaryotes and
`Function as Nucleolar Organizers
`443
`Small Nucleolar RNAs (snoRNAs)Assist in Processing
`tRNAs and Assembling Ribosome Subunits
`444
`Self-Splicing Group I Introns Were the First Examples of
`Catalytic RNA 445
`All Pre-tRNAs Undergo Cleavage and Base
`Modification
`446
`
`

`

`
`
`|
`
`T1
`|1
`
`||
`
`Contents | xxv
`
`DNA Damage Can Be Repaired by Several
`Mechanisms
`475
`Euka