MOLECULAR BIOLOGY OF
`
`THIRD EDITION
`
`Bruce Alberts • Dennis Bray
`Julian Lewis • Martin Raff• Keith Roberts
`James D. Watson
`
`Garland Publishing, Inc.
`New York& London
`
`1 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`Text Editor: Miranda Hobertson
`Managing Editor: Ruth Adams
`Illustrator: Nigel Orme
`Molecular Model Drawings: Kale Hesketh-Moore
`Director of Electronic Publishing: John M-Hoblin
`Computer Specialist: Chuck Bartelt
`Disk Preparation: Carol Winter
`Copy Editor: Shirley M. Cobert
`Production Editor: Douglas Goertzen
`Production Coordinator: Perry Bessas
`Indexer: Maija Hinkle
`
`Bruce Alberts received his Ph.D. from Harvard University and is
`currently President of the National Academy of Sciences and Professor
`of Biochemistry and Biophysics at the University of California, San
`Francisco. Dennis Bray received his Ph.D. from the Massachusetts
`Institute of Technology and is currently a Medical Research Council
`Fellow in the Depanment of Zoology, University of Cambridge.
`}11/ian Lewis received his D.Phil. from the University of Oxford and is
`currently a <)enior Scientisl in the Imperial Cancer Research Fund
`Developmental Biology Unit, University of Oxford. Martin Raflreceived
`his M.D. from McGill University and is currently a Professor in the M HC
`Laboratory for Molecular Cell Biology and the Biology Depal'lment,
`University College London. Keith Roberts received his Ph.D. from the
`University of Cambridge and is currently Head of the Department of Cell
`Biology, the John Innes l nslitute, Norwich. James D. Watson received his
`Ph.D. from Indiana University and is currently Director of the Cold Spring
`Harbor Laboratory. He is t11e a u thor of lvlolecular Biology of the Gene and,
`with Francis Crick and Maurice Wilkins, won the Nobel Prize in Medicine
`and Physiology in 1962.
`
`© 1983, 1989, 1994 by Bruce Alberts, Dennis Bray, Julian Lewis,
`Martin Haff, Keith Roberts, and James D. Watson.
`
`All rights reserved. No part of this book covered by the copyright hereon
`may be reproduced or used in any form or by any means-graphic,
`electronic, or mechanical, including photocopying, recording, taping, or
`information storage and retrieval systems-without permission of the
`publisher.
`
`Libra ry of Con gress Cataloging-in-Pub licatio n Data
`Mole..:ular biology of the cell / Bruce Alberts ... let al.).-3rd ed.
`p. cm.
`Includes bibliographical references and index.
`ISBN 0-8153- 1619-4 {hard cover).-
`ISBN 0-8153-1620-8 (pbk.)
`l. Cytology. 2. Molecular biology. I. Alberts, Bruce.
`(DNLM: l. Cells. 2. Molecular J3iolog~1. QI-I 581.2 M718 1994]
`QH58l.2.M64 1994
`574.87-dc20
`DNLM/DLC
`for Libraty of Congress
`
`93-45907
`CrP
`
`Published by Garland Publishing, Inc.
`717 Fifth Avenue, New York, NY 10022
`
`Printed in the United States of America
`15 14 13 12 10 9 8 7 6 5 4
`
`Fron t cover: The photograph shows a rat nerve cell
`in culture. It is labeled (yellow) with a lluorescent
`antibody that stains its cell body and clendritic
`processes. Nerve terminals (green} from other
`neurons (not visible}, which have made synapses on
`the cell, are labeled with a different antibody.
`(Courtesy of Olaf Mundigl and Pietro de Camilli.}
`Dedication page: Gavin Borden. late president
`of Garland Publishing. weathered in during his
`mid- l980s climb near Mount McKinley with
`M BoC author Bruce Alberts and famous mountaineer
`guide Mugs Stump (1940- 1992).
`Back cover: The authors, in alphabetical order,
`crossing Abbey Road in London on their way to lunch.
`Much of this third edition was written in a house just
`arnund the corner. (Photograph by Richard Olivier.)
`
`2 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`List of Topics
`
`Introduction to the Cell
`
`The Evolution of the Cell
`
`4
`
`4
`
`Frnm Molecules to the First Cell
`Simple Biological Molecules Can Form Under
`Prebiotic Conditions
`Complex Chemical Systems Can Develop
`in an Environment That ls Far from
`Chemical Equilibrium
`Polynucleotides Are Capable of Directing
`Their Own Synthesis
`Self-replicating Molecules Undergo Natural Selection
`Specialized RNA Molecules Can Catalyze
`Biochemical Reactions
`7
`Information Flows from Polynucleotides to Polypeptides
`9
`Membranes Defined tbe First Cell
`9
`All Present-Day Cells Use DNA as Their Hereditary Material 10
`Summmy
`11
`
`4
`
`5
`7
`
`From Procaryo tes to Eucaryotes
`Procaryotic Cells Are Structurally Simple
`but Biochemically Diverse
`Metabolic Reactions Evolve
`Evolutionary Relationships Can Be Deduced
`by Comparing ONA Sequences
`Cyanobacteria Can Fix C02 and N2
`Bacteria Can Carry Out the Aerobic Oxidation
`of Food Molecules
`Eucaryotic Cells Con tain Several Distinctive Organelles
`Eucaryotic Cells Depend on Mitochondria
`for Their Oxidative Metabolism
`Chloroplasts Are Lhe Desc.enuants of an Engulfed
`Procaryotic Cell
`
`12
`
`L2
`13
`
`14
`15
`
`16
`17
`
`20
`
`21
`
`Part
`I
`
`Chapter
`l
`
`Eucaryotic Cells Contain a Rich Array
`of Internal Membranes
`Eucaryotic Cells Have a Cytoskeleton
`Protozoa Include the Most Complex Cells Known
`Tn Eucaryotic Cells the Genetic Material Is Packaged
`in Complex Ways
`S11111mmy
`
`From Single Cells to Multicellu lar Organisms
`Single Cells Can Associate to Form Colonies
`The Cells of a Higher Organism Become Specialized
`and Cooperate
`Multicellular Organization Depends on Cohesion
`Between Cells
`Epithelial Sheets of Cells Enclose a Sheltered
`Internal Environmelll
`Cell-Cell Communication Controls the Spatial Pattern
`of Multicellular Organisms
`Cell Memory Permits the Development
`of Complex Patterns
`Basic Developmental Programs Tend to Be Conserved
`in Evolution
`The Cells of the Vertebrate Body Exhibit More Than
`200 Different Modes of Specialization
`Genes Can Be Switched On and Off
`Sequence Comparisons Reveal Hundreds of Families
`of I Iomologous Genes
`Summa1y
`
`H.eferences
`
`22
`23
`24
`
`25
`25
`
`26
`26
`
`27
`
`30
`
`30
`
`31
`
`32
`
`32
`
`34
`34
`
`35
`39
`
`39
`
`Small Molecules, Energy, and Biosynthesis
`
`The Ch emical Components of a Cell
`Cell Chemistry Ts Based on Carbon Compounds
`Cells Use Four Basic Types of Small Molecules
`Sugars Are Food Molecules of the Cell
`Fatty Acids Are Components of Cell Membranes
`Amino Acids Are the Subunits of Proteins
`Nucleotides Are the Subunits of ONA and RNA
`Su 111111 my
`
`4 1
`41
`43
`43
`45
`46
`46
`60
`
`Biological Ord er and En ergy
`Biological Order ls Made Possible by the Release
`of Heal Energy from Cells
`Photosynthetic Organisms Use Sunlight to Synthesize
`Organic Compounds
`Chemical Energy Passes from Plants to Animals
`Cells Obtain Energy by the Oxidation
`of Biological Molecules
`The Breakdown of an Organic Molecule Takes Place
`in a Sequence of Enzyme-catalyzed Reactions
`
`Chapter
`2
`
`60
`
`60
`
`61
`61
`
`62
`
`63
`
`xvii
`
`3 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`Part of the Energy Released in OxidaLion Reactions
`Is Coupled to the Formation of ATP
`The Hydrolysis of ATP Generates Order in Cells
`S II Ill 111 my
`
`Food and the Derivation of Cellular Energy
`Food Molecules Are Broken Down in Three Stages
`LO Give ATP
`Glycolysis Can Produce ATP Even in the Absence ·
`ofO:-.')'gcn
`NADH Is a Central Intermediate in Oxidative Catabolism
`Metabolism Is Dominated b}' U1e Citric Acid Cycle
`In Oxidative Phosphorylation the Transfer of Electrons
`10 Oxygen Drives ATP Formation
`Amino Acids and Nucleotides Are Part
`of the Nitrogen Cycle
`Summmy
`
`Biosynthesis and the Creation of Order
`The Free-Energy Change for a Reaction Determines
`Whether Il Can Occur
`Biosynthetic Reactions Are Often Directly Coupled
`LO ATP Hydrolysis
`
`64
`65
`66
`
`66
`
`66
`
`68
`70
`71
`
`72
`
`73
`74
`
`74
`
`74
`
`75
`
`Coenzymes Are Involved in the Transfer of Specific
`Chemical Groups
`The Structure of Coenzymes Suggests That They May
`Have Originated in an RNA World
`Biosynthesis Requires Hcducing Power
`Biological Polymers Arc Synthesized by Repetition
`of Elementa1y Dehydration Reactions
`s 11111111(/1)1
`The Coordination of Catabolism
`and Biosynthesis
`Metabolism Is Organized and Regulated
`Metabolic Pathways Are Regulated by Changes
`in Enzyme Activity
`Catabolic Reactions Can Be Reversed by an Input
`of Energy
`Enzymes Can Be Switched On and Off
`by Covalent Modification
`Reactions Are Compartmentalized Both Within Cells
`and Within Organisms
`Swnmruy
`
`References
`
`77
`
`78
`79
`
`80
`81
`
`82
`82
`
`82
`
`82
`
`86
`
`86
`87
`
`88
`
`Macro1nolecules: Structure, Shape, and lnforn1ation
`
`Chapter
`3
`
`tRNA Molecules Match Amino Acids to Groups
`of Nucleotides
`The RNA Message Is Read from One End to the Other
`by a Ribosome
`Some RNA Molecules Function as Catalysts
`S II Ill llW I)'
`
`106
`
`107
`108
`110
`
`111
`
`111
`
`Protein Structure
`The Shape of a Protein Molecule ls Determined
`by Its Amino Acid Sequence
`Common Folding Pallerns Recur in Different
`113
`Protein Chains
`115
`Proteins Are Amazingly Versatile Molecules
`Proteins Have Different Levels of Structural Organization 116
`Domains Arc Formed from a Polypeptide Chain
`Thar Winds Back and Forth, Making Sharp Turns
`at the Protein Surface
`Relatively Few of the Many Possible Polypeptide
`Chains Would Be Useful
`New Proteins Usually Evolve by Alterations of Old Ones
`New Proteins Can Evolve by Recombining Preexisting
`Polypeptide Domains
`Structural Homologies Can Help Assign Functions
`to Newly Discovered Proteins
`Protein Subunits Can Assemble into Large s,ructurcs
`A Single Type of Protein Subun it Can Interact with Itself
`to Form Geometrically Regular Assemblies
`Coiled-Coil Proteins Help Build Many Elongated
`Structures in Cells
`Proteins Can Assemble into Sheets, Tubes, or Spheres
`
`118
`
`119
`120
`
`121
`
`122
`123
`
`12'1
`
`124
`12,5
`
`Molecular Recognition Processes
`The Specific Interactions of a Macromolecule Depend
`on Weak, Noncovalent Bonds
`A Helix ls a Common Structural Motif in Biological
`Structures Made from Repeated Subunits
`Diffusion Is the First Step to Molecular Recognition
`Thermal Motions Bring Molecules Together and Then
`Pull Them Apart
`The Equilibrium Constant Is a Measure of U1e Strength
`of an Interaction Between Two Molecules
`Atoms and Molecules Move Very Rapidly
`Molecular Recognition Processes Can Never Be Perfect
`S 11111111 my
`
`Nucleic Acids
`Genes Are Made of DNA
`DNA Molecules Consist of1\vo Long Chains Held
`Together b)• Complementaiy Base Pairs
`The Structure of DNA Provides an Explanation
`for Heredity
`Errors in DNA Replication Cause Mutations
`The Nucleotide Sequence of a Gene Determines
`the Amino Acid Sequence of a Protein
`Portions of DNA Sequence Are Copied into RNA
`Molecules That Guide Protein Synthesis
`Eucaryolic RNA Molecules Are Spliced to Remove
`lntron Sequences
`Sequences of Nucleotides in mRNA Are "Read'' in Sets
`ofTluee and Translated into Amino Acids
`
`89
`
`90
`
`94
`95
`
`95
`
`96
`97
`97
`98
`
`98
`98
`
`98
`
`99
`102
`
`104
`
`104
`
`105
`
`106
`
`xviii
`
`Lis t of Topics
`
`4 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`Many Structures in Cells Are Capable of Self-assembly
`Not /\II Biological Structures Form by Self-assembly
`S 11111111 my
`
`Proteins as Catalysts
`A Protein's Conformation Determines Its Chemistry
`Substrate Binding ls the Pil'st Step in Enzyme Catalysis
`Enzymes Speed Reactions by Selectively Stabilizing
`· Transition States
`Enzymes Can Promote the Making and Breaking
`of Covalent Bonds Through Simultaneous Acid
`and Base Catalysis
`
`How Cells Are Studied
`
`Looking at the Structw·e of Cells
`in the Microscope
`The Light Microscope Can Resolve Details 0.2 µm Apart
`Tissues Are Usually Fixed and Sectioned
`for Microscopy
`Different Components of the Cell Can
`Be Selectively Stained
`Specific Molecules Can Be Located in Cells
`by Fluorescence Microscopy
`Living Cells Are Seen Clearly in a Phase-Contrast
`or a Differential-Interference-Contrast Microscope
`Images Can Be Enhanced and Analyzed
`by Electronic Techniques
`Imaging of Complex Three-dimensional Objects
`Is Possible with the Confocal Scanning Microscope
`The Electron Microscope Resolves the Fine Structure
`of the Cell
`Biological Specimens Require Special Preparation
`for the Electron Microscope
`Three-dimensional Images of Surfaces Can Be Obtained
`by Scanning Electron Microscopy
`Metal Shadowing Allows Surface Features
`to Be Examined at High Resolution
`by Transmission Electron Microscopy
`Freeze-Fracture and Freeze-Etch Electron Microscopy
`Provide Unique Views of the Cell Interior
`Negative Staining and C1yoelectron Microscopy Nlow
`Macromolecules to Be Viewed at High Resolution
`511111111(//y
`
`Isolating Cells and Growing Them in Culture
`Cells Can Be Isolated from a Tissue and Separated
`into Different Types
`Cells Can Be Grown in a Culture Dish
`Serum-free, Chemically Defined Media Permit
`Identification of Specific Growth Factors
`Eucaryotic Cell Lines Are a Widely Used Source
`of Homogeneous Cells
`Cells Can 13e Fused Together to Form Hybrid Cells
`$11111111 aty
`
`127
`128
`128
`
`128
`129
`130
`
`131
`
`132
`
`139
`141
`
`142
`
`143
`
`144
`
`145
`
`146
`
`147
`
`148
`
`150
`
`152
`
`153
`
`153
`
`154
`156
`
`156
`
`157
`158
`
`159
`
`160
`160
`162
`
`List of Topics
`
`Enzymes Can Further Increase Reaction Rates
`by Forming Covalent Intermediates
`with Their Substrates
`Enzymes Accelerate Chemical Reactions but Cannot
`Make Them Energetically More Favorable
`Enzymes Determine Reaction Paths by Coupling
`Selected Reactions to ATP Hydrolysis
`Multienzyme Complexes Help to Increase the Hate
`of Cell Metabolism
`Sl/lnmaty
`
`References
`
`133
`
`133
`
`133
`
`134
`135
`
`136
`
`Chapter
`4
`
`Fractionation of Cells and Analysis of Their
`Molecules
`Organelles and Macromolecules Can Be Separated
`by Ultracentrifugation
`The Molecular Details of Complex Cellu lar Processes
`Can Be Deciphered in Cell-free Systems
`Proteins Can Be Separated by Chromatography
`The Size and Subunit Composition of a Protein
`Can Be Determined by SOS Polyac1ylamide·
`Gel Electrophoresis
`More Than 1000 Proteins Can Be Resolved on
`a Single Gel by Two-ctimensional Polyacrylamide-
`Gel Electrophoresis
`Selective Cleavage of a Protein Generates a Distinctive
`Set of Peptide Fragments
`Short Amino Acid Sequences Can Be Analyzed
`by Automated Machines
`The Diffraction of X-rays by Protein Crystals Can Reveal
`a Protein's Exact Structure
`Molecular Structure Can Also Be Determined Using
`Nuclear Magnetic Resonance {NMR) Spectroscopy
`Summmy
`
`162
`
`162
`
`164
`166
`
`169
`
`170
`
`173
`
`173
`
`174
`
`175
`176
`
`178
`Tracing and Assaying Molecules Inside Cells
`Radioactive Atoms Can Be Detected with Great Sensitivity 178
`Radioisotopes Are Used to Trace Molecules in Cells
`and Organisms
`Ion Concentrations Can Be Measured with
`Intracellular Electrodes
`Rapidly Changing Intracellular Jon Concentrations Can
`Be Measured with Light-emitting Indicators
`There Are Several Ways of Introducing Membrane-
`impermeant Molecules into Cells
`The Light-induced Activation of "Caged" Precursor
`MolecLtles Facilitates Studies of Intracellular Dynamics 184
`Antibodies Can Be Used to Detect and Isolate Specific
`Molecules
`1-Iybridoma Cell Lines Provide a Permanent Source
`of Monoclonal Antibodies
`Su111111aiy
`
`178
`
`181
`
`182
`
`183
`
`186
`
`187
`189
`
`References
`
`189
`
`xix
`
`5 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`Molecular Genetics
`
`Protein Function
`
`Part
`II
`
`Chapter
`5
`
`Making Mach ines Ou t of P roteins
`The Binding of a Ligand Can Change the Shape
`of a Protein
`Two Ligands That Bind to the Same Protein Often
`Affect Each Other's Binding
`Two Ligands Whose Binding Sites Are Coupled Must
`Reciprocally Affect Each Other's Binding
`Allosteric Transitions Help Regulate Metabolism
`Proteins orten Form Symmetrical Assemblies That
`Undergo Cooperative Atlosteric Transitions
`The Allosteric Transition in Aspartale Transcarbamoylase
`ls Understood in Atomic Detail
`Protein Phospho1ylation ls a Common Way of Driving
`Allosteric Transitions in Euca1yotic Cells
`A Eucaiyotic Cell Contains Many Protein Kinases and
`Phosphatases
`The Structure of Cdk Protein Kinase Shows How
`a Protein Can Function as a Microchip
`Proteins That Bind and Hydrolyze GTP Are Ubiquitous
`Cellular Regulators
`Other Proteins Control the Activity of GTP-binding
`Proteins by Determining Whether GTP
`or GDP Is Bound
`The AUosteric Transition in EF-Tu Shows How Large
`Movements Can Be Generated from Small Ones
`Proteins That Hydrol)rze ATP Do Mechanical
`Work in Cells
`The Structure of Myosin Reveals How Muscles
`Exert Force
`
`195
`
`195
`
`196
`
`197
`198
`
`199
`
`200
`
`202
`
`20<!
`
`204
`
`206
`
`206
`
`207
`
`208
`
`209
`
`ATP-driven Membrane-bound Allosteric Proteins
`Can Either Act as Ion Pumps or Work in Heverse
`to Synthesize ATP
`Energy-coupled Allosteric Transitions in Proteins Allow
`the Proteins to Function as Motors, Clocks, Assembl)'
`Factors, or Transducers of Information
`Proteins Often Form Large Complexes That Function
`as Protein Machines
`Summary
`
`The BiJ:th , Assembly, an d Death of Protein s
`Proteins Are Thought to Fold Through a Molten
`Globule Intermediate
`Molecular Chaperones Help Guide the Folding
`ofa Protein
`Many Proteins Contain a Series of Independently
`folded Modules
`Modules Confer Versatility and Often Mediate
`Protein-Protein Interactions
`Proteins Can Bind to Each OLher Through Several
`Types oflnterfaces
`Linkage and Selective Proteolysis Ensure All-or-None
`Assembly
`Ubiquitin-dependent Proteolytic Pathways
`Are Largely Responsible for Selective Protein
`Turnover in Eucruyotes
`The Lifetime of a Protein Can Be Determined
`by Enzymes That Alter Its N-Terminus
`Summaiy
`
`References
`
`209
`
`211
`
`211
`212
`
`212
`
`213
`
`214
`
`215
`
`216
`
`217
`
`217
`
`218
`
`220
`22l
`
`221
`
`The Binding of Many Hibosomes to an Individual
`mRNA Molecule Generates Polyribosomes
`The Overall Hate of Protein Syn! hes is in Eucaryotes
`Is Controlled by Initiation Factors
`The Fidelity of Protein Synthesis Is Improved
`by Two Proofreading Mechanisms
`Man)' Inhibitors of Procaryotic Protein Synthesis
`Are Useful as Antibiotics
`I-low Did Protein Synthesis Evolve?
`Summmy
`
`Chapter
`6
`
`237
`
`238
`
`239
`
`240
`241
`242
`
`Basic Genetic Mechanisms
`
`RNA and Protein Synthesis
`RNA Polymerase Copies DNA into RNA: The Process
`of DNA Transcription
`Only Selected Portions of a Chromosome Are Used
`to Produce HNA Molecules
`Transfer RNA Molecules Act as Adaptors Thal Translate
`Nucleotide Sequences into Protein Sequences
`Specific Enzymes Couple Each Amino Acid
`to Its Appropriate tRNA Molecule
`Amino Acids Are Added to the Carboxyl-Terminal End
`of a Growing Polypeptide Chain
`The Genetic Code Is Degenerate
`The Events in Protein Synthesis Are Catalyzed
`on the Ribosome
`A Ribosome Moves Stepwise Along the mRNA Chain
`A Protein Chain ls Released from the Hibosome When
`Any One of Three Stop Codons Is Reached
`The Initiation Process Sets the Reading Frame
`for Protein Synthesis
`Only One Species of Polypeptide Chain Is Usually
`Synthesized from l::ach mRNA Molecule in Eucaryotes
`
`223
`
`224
`
`226
`
`227
`
`228
`
`230
`230
`
`231
`232
`
`234
`
`234
`
`236
`
`xx List of Topics
`
`DNA Repair
`DNA Sequences Are Maintained with Very High Fidelity
`The Observed Mutation Hates in Proliferating Cells
`Arn Consistent with Evolutionary Estimates
`Most Mutations in Proteins Are Deleterious
`and Are Eliminated by Natural Selection
`243
`Low. Mutat ion Rates Are Necessary for Life as We Know It 243
`Low Mucalion Rates Mean That Related Organisms Must
`Be Made from Essentially the Same Proteins
`
`242
`242
`
`243
`
`244
`
`6 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`If Left Uncorrected, Spontaneous DNA Damage Would
`Rapidly Change DNA Sequences
`The Stability of Genes Depends on DNA Repair
`DNA Damage Can Be Removed by More Than
`One Pathway
`Cells Can Produce DNA Repair Enzymes in Response
`to DNA Damage
`The Structure and Chemistry of the DNA Double Helix
`Make It Easy to Hepair
`511111 mc11y
`
`DNA Replication
`Base-pairing Underlies DNA Replication as well as
`DNA Repair
`The DNA Replication Fork Is Asymmetrical
`The l ligh Fidelity of DNA Replication Requires
`a Proofreading Mechanism
`Only DNA Replication in the 5'-to-3' Direction Allows
`Efficient Error Correction
`A Special Nucleotide Polymerizing Enzyme Synthesizes
`Short RNA Primer Molecules on Lhe Lagging Strand
`Special Proteins Help Open Up the DNA Double Tlelix
`in Front of the Replication Fork
`A Moving DNA Polymerase Molecule Is Kept Tethered
`to the DNA by a Sliding Ring
`The Proteins ar a Replicarion Fork Cooperate to Form
`a Replication Machine
`A Mismatch Proofreading System Removes Replication
`Errors That Escape from the Replication Machine
`Replication Forks Initiate at Replication Origins
`DNA Topoisomerases Prevent DNA Tangling
`During Replication
`DNA Replication Is Basically Similar in Eucaiyotes
`and Procaryotes
`Sum mat)'
`
`Genetic Recombination
`General Recombination Is Guided by Base-pairing
`Interactions Between Complementary Strands
`of Two Homologous DNA Molecules
`General Recombination Can Be Initiated at a Nick
`in One Strand of a DNA Double Helix
`DNA Hybridization Reactions Provide a Simple Model
`for the Base-pairing Step in General Recombination
`
`244
`245
`
`247
`
`249
`
`250
`251
`
`251
`
`251
`252
`
`253
`
`254
`
`254
`
`255
`
`256
`
`256
`
`258
`259
`
`260
`
`262
`262
`
`263
`
`263
`
`264
`
`265
`
`The RecA Protein Enables a DN1\ Single Strand
`to Pair wiLh a Homologous Region of DNA
`Double Helix in£. coli
`General Genetic Recombination Usually Involves
`a Cross-Strand Exchange
`Gene Conversion Results rrom Combining General
`Recombination and L.imited DNA Symhesis
`Mismatch Proofreadi ng Can Prevent Promiscuous
`Genetic Recombination Between Two Poorly
`Matched DNA Sequences
`Site-specific Recombination Enzymes Move Special
`DNA Sequences into and out of Genomes
`Transpositional Recombination Can Insert a Mobile
`Genetic Element into Any DNA Sequence
`S LI 111111 my
`
`Viruses, Plasmids, and Transposable
`Genetic Elements
`Viruses Are Mobile Genetic Elements
`The Outer Coat of a Virus May Be a Protein Capsid
`or a Membrane Envelope
`Viral Genomes Come in a Variety of Forms
`and Can Be Either RNA or DNA
`A Viral Chromosome Codes for Enzymes Involved
`in the Replication of lts Nucleic Acid
`Both RNA Viruses and DNA Viruses Replicate Through
`the Formation of Complementary Strands
`Viruses Exploit the Intracellular Traffic Machinery
`of Their Ilost Cells
`Different Enveloped Viruses Bud from Different
`Cellular Membranes
`Viral Chromosomes Can Integrate
`into Host Chromosomes
`The Continuous Synthesis of Some Viral Proteins
`Can Make Cells Cancerous
`RNA Tumor Viruses Are Retroviruses
`The Virus That Causes AIDS ls a Retrovirus
`Some Transposable Elements Are Close Relatives
`of Retroviruses
`Other Transposable Elements Transfer Themselves
`Directly from One Site in the Genome to Another
`Most Viruses Probably Evolved from Plasmids
`S LI 111111 GIJI
`
`References
`
`266
`
`268
`
`268
`
`270
`
`271
`
`272
`273
`
`273
`274
`
`2U
`
`276
`
`277
`
`277
`
`278
`
`280
`
`281
`
`282
`282
`284
`
`284
`
`285
`286
`287
`
`287
`
`Recombinant DNA Technology
`
`Chapter
`7
`
`The Fragmentation, Separation,
`and Sequencing of DNA Molecul es
`Restriction Nucleases Hydrolyze DNA Molecules
`at Specific Nucleotide Sequences
`Restriction Maps Show the Distribution of Short Marker
`Nucleotide Sequences Along a Chromosome
`Gel Electrophoresis Separates DNA Molecules
`of Different Sizes
`Purified DNA Molecules Can Be Specifically Labeled
`with Radioisotopes or Chemical Markers in Vitro
`Isolated DNA Fragments Can Be Rapidly Sequenced
`DNA Footprinting Reveals the Sites Where Proteins
`Bind on a DNA Molecule
`S11mma1)1
`
`292
`
`293
`
`293
`
`293
`
`295
`296
`
`297
`298
`
`Nucleic Acid Hybridization
`Nucleic Acid Hybridization Reactions Provide a Sensitive
`Way of Detecting Specific Nucleotide Sequences
`Northern and Sou thern Blotting Facilitate
`Hybridization with Electrophoretically Separated
`Nucleic Acid Molecules
`RFLP Markers Greatly Facilitate Genetic Approaches
`to the Mapping and Analysis of Large Genomes
`S}mthetic DNA Molecules Facilitate the Prenatal
`Diagnosis of Genetic Diseases
`Hybridization at Reduced Stringency Allows Even
`Distantly Related Genes to Be Identified
`In Silll I lybridization Techniques Locate Specific
`Nucleic Acid Sequences in Cells or on Chromosomes
`S 11111111 my
`
`List of Topics
`
`300
`
`300
`
`30 I
`
`303
`
`305
`
`306
`
`307
`307
`
`xxi
`
`7 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`DNA Cloning
`A DNA Librmy Can Be Made Using Either Viral
`or Plasmid Vectors
`Two Types of DNA Libraries Serve Different Purposes
`cDNA Clones Contain Uninterrupted Coding Sequences
`cDNA Libra1'ics Can Be Prepared from Selected
`Populations of mRNA Molecules
`Either a DNA Probe or a Test for Expressed Protein
`Can Be Used to Identify the Clones of Interest
`in a DNA Library
`In Vitro Translation Facilitates Identification
`of the Correct DNA Clone
`The Selection of Overlapping DNA Clones Allows One
`to "Walk" Along the Chromosome to a Nearby
`Gene of Interest
`Ordered Genomic Clone Libraries Are Being Produced
`for Selected Organisms
`Positional DNA Cloning Reveals Human Genes
`with Unanticipated Functions
`Selected DNA Segments Can Be Cloned in a Test Tube
`by a Polymerase Chain Reaction
`S II 117 Ill Cl1J'
`
`308
`
`308
`309
`310
`
`311
`
`312
`
`313
`
`314
`
`314
`
`315
`
`316
`318
`
`DNA Engineering
`New DNA Molecules of Any Sequence Can Be Formed
`by Joining Together DNA Fragments
`
`319
`
`319
`
`Homogeneous RNA Molecules Can Be Produced
`in Large Quantities by DNA Transcription in \litro
`Rare Cellular Proteins Can Be Made in Large Amounts
`Using Expression Veccors
`Reporter Genes Enable Regulatory DNA Sequences
`to 13e Dissected
`Mutant Organ isms Best Reveal the Funclion of a Gene
`Cells Containing Mutated Genes CmJ Be Made to Order
`Genes Can Be Redesigned to Produce Proteins
`of Any Desired Sequence
`Fusion Proteins Are Often Useful for Analyzing
`Protein Function
`Normal Genes Can Be Easily Replaced by Mutant Ones
`in Bacteria and Some Lower Euca1yoles
`Engineered Genes Can Be Used to Create Specific
`Dominant Mutations in Diploid Organ isms
`Engineered Genes Can Be Permanently Inserted into
`the Germ Line of Mice or Fruit Flies to Produce
`Transgenic Animals
`Gene Targeting Makes It Possible lo Produce
`Trm1sgenic Mice Thal Are Missing Specific Genes
`Transgenic Plants Are Important for Both Cell Biology
`and Agriculture
`S11m mmr
`
`References
`
`320
`
`320
`
`321
`321
`323
`
`323
`
`324
`
`325
`
`326
`
`327
`
`329
`
`330
`330
`
`332
`
`The Cell Nucleus
`
`Chromosomal DNA and Its Packaging
`Each DNA Molecule !hat Forms a Linear Chromosome
`Must Contain a Centromere, Two Telomeres,
`and Replication Origins
`Most Chromosomal DNA Does Not Code
`for Proteins or RNAs
`Each Gene Produces an RNA MolecuJe
`Comparisons Between the DNAs of Related Organisms
`Distinguish Conserved and Nonconserved Regions
`of DNA Sequence
`1-Iistones Are the Principal Structural Proteins
`of Eucaryotic Chromosomes
`Histones Associate with DNA to Form Nucleosomes,
`the Unit Particles of Chromatin
`The Positioning of Nucleosomes on DNA Ts Determined
`by U1e Propen sity of the DNA to Form Tight Loops
`and by the Presence of Other DNA-bound Proteins
`Nucleosomes Are Usually Packed Together by llistone
`Hl to Form Regular Higher-Order Structures
`S111nma1y
`
`The Global Structure of Chromosomes
`Lampbrush Cluomosomes Contain Loops
`ofDecondensed Chromatin
`Orderly Domains of Interphase Chromatin Also
`Can Be Seen in Insect Polytene Chromosomes
`Individual Chromatin Domains Can Unfold
`and Refold as a Unit
`Both Bands and Interbands in Polytene Chromosomes
`Are Likely to Contain Genes
`Transcriplionally Active Chromatin Is Less Condensed
`Active Chromatin Is Biochemically Distinct
`
`336
`
`337
`
`339
`340
`
`341
`
`342
`
`342
`
`344
`
`345
`346
`
`346
`
`346
`
`348
`
`349
`
`350
`351
`352
`
`Chapter
`8
`
`Heterocluomatin Is Highly Condensed
`and Transcriprionally Inactive
`Mitotic Chromosomes Are Formed from Chromatin
`in Its Most Condensed State
`Each Mi totic Chromosome Contains a Characteristic
`Pattern of Very Large Domains
`Sum maiy
`
`Chromosome Replication
`Specific DNA Sequences Serve as Replication Origins
`A Mammalian Cell-free System Replicates
`the Chromosome of a Monkey Virus
`Replication Origins Are Activated in Clusters
`on 1-Ugher Eucruyotic Chromosomes
`Different Regions on the Same Chromosome Replicate
`at Distinct Times
`Highly Condensed Chromatin Heplicates Late,
`While Genes in Active Chromatin Replicate Early
`The Lale-replicating Replication Units Coincide with
`the A-T-rich Bands on Metaphase Chromosomes
`The Controlled Timing of DNA Replication May
`Contribute to Cell Memory
`Chromatin-bow1d Factors Ensure That Each Region
`of the DNA Is Replicated Only Once
`New Histones Are Assembled into Chromatin
`as DNA Replicates
`Telomeres Consist of Short G-rich Repeats That
`Arc Added to Chromosome Ends by Telomerase
`S LI 111 Ill my
`
`RNA Synthesis and RNA Processing
`RNA Polymerase Exchanges Subunits as It Begins
`Each RNA Chain
`
`353
`
`353
`
`355
`355
`
`356
`357
`
`358
`
`359
`
`360
`
`360
`
`361
`
`361
`
`362
`
`363
`
`364
`365
`
`365
`
`365
`
`xxii
`
`List of Topics
`
`8 of 64
`
`Celltrion, Inc., Exhibit 1005
`
`

`

`367
`
`368
`
`371
`
`372
`
`373
`
`374
`
`375
`
`375
`
`376
`
`Three Kinds of RNA Polymerase Make RNA in Eucaryotes 366
`RNA Polymerase 11 Transcribes Some DNA Sequences
`Much More Often Than Others
`The Precursors of Messenger RNA Are Covalently
`Modified at Both Ends
`RNA Processing Removes Long Nucleotide Sequences
`from the Middle of RNA Molecules
`hnRNA Transcripts Are fmmediately Coated
`· with Proteins and snRNPs
`lntron Sequences Are Removed as Lariat-shaped
`RNA Molecules
`Multiple lntron Sequences Arc Usually Removed
`from Each RNA Transcript
`Studies ofThalassemia Reveal How RNA Splicing
`Can Allow New Proteins to Evolve
`Spliceosome-catalyzed RNA Splicing Probably Evolved
`from Self-splicing Mechanisms
`The Transport of mRNAs to the Cytoplasm Is Delayed
`Until Splicing Is Complete
`Ribosomal RNAs (rRNAs} Are Transcribed
`from Tandemly Arranged Sets of Identical Genes
`The Nucleolus ls a Ribosome-producing lVlachine
`The Nucleolus ls a Highly Orgru1ized Subcompartment
`of the Nucleus
`The Nucleolus Is Reassembled on Specific
`Chromosomes After Each Mitosis
`Individual Chromosomes Occupy Discrete Territories
`in the Nucleus During Interphase
`How Well Ordered ls the Nucleus?
`
`378
`379
`
`381
`
`382
`
`383
`384
`
`Control of Gene Expression
`
`4 01
`
`An Overview of Gene Con tro l
`The Different Cell Types of a Multicellular Organism
`Contab1 the Same DNA
`401
`Different Cell Types Synthesize Different Sets of Proteins 402
`A Cell Can Chru1ge the Expression of Its Genes
`in Response to External Signals
`Gene Expression Can Be Regulated at Many of the Steps
`in the Pathway from DNA to RNA to Protein
`Su111111my
`
`403
`
`403
`404
`
`DNA-bindi ng M otifs in Gene
`Regulatory Proteins
`Gene Regulatory Proteins Were Discovered Using
`Bacterial Genetics
`The Outside of the DNA Helix Can Be Read by Proteins
`The Geometry of the DNA Double Helix Depends
`on the Nucleotide Sequence
`Short DNA Sequences Are Fundamental Components
`of Genetic Switches
`Gene Regulato1y Proteins Contain Structural Motifs
`That Can Read DNA Sequences
`The Helix-Tum-Helix Motifls One of the Simplest
`and Most Common DNA-binding Motifs
`Homeodomain Proteins Are a Special Class
`of Helix-Turn-Helix Proteins
`There Are Several Types of DNA-binding
`Zinc Finger Molifs
`P Sheets Can Also Recognize DNA
`The Leucine Zipper Motif Mediates Both DNA Binding
`and Protein Dimerizalion
`
`404
`
`404
`405
`
`406
`
`407
`
`408
`
`408
`
`410
`
`410
`412
`
`412
`
`List of Topics
`
`S11mma1y
`
`The Organization and Evolution
`of the NucJear Genome
`Genomes Are Fine-tuned by Point M~tation
`and Radically Remodeled or Enlarged
`by Genetic Hecombination
`Tandem ly Repeated DNA Sequences Tend
`to Remain the Same
`The Evolution of the Glob in Gene Family Shows
`How Random DNA Duplications Contribute
`to the Evolution of Organisms
`Genes Encoding New Proteins Can Be Created
`by the Recombination of Exons
`Most Proteins Probably Originated from
`Highly Split Genes
`A Major Fraction of the DNA of Higher Eucaryotes
`Consists of Repeated, Noncoding
`Nucleotide Sequences
`Satellite DNA Sequences Have No Known Function
`The Evolution of Genomes Has Been Accelerated
`by Transposable Elements
`Transposable Elements Often Affect Gene Regulation
`Tra.nsposition Bursts Cause Cataclysmic Changes
`111 Genomes and Increase Biological Diversity
`Aboul 10% of the Human Genome Consists
`of Two Families of Transposable Elements
`S LI 111111 my
`
`Referen ces
`
`385
`
`385
`
`386
`
`386
`
`387
`
`389
`
`389
`
`391
`391
`
`392
`393
`
`394
`
`395
`395
`
`396
`
`Chapter
`9
`
`The Helix-Loop-Helix Motif Also Mediates
`Dimerization and DNA Binding
`It ls Not Yet Possible to Predict the DNA Sequence
`Recognized by a Gene Regulatory Protein
`A Gel-Mobility Shift Assay Allo