Cee ey
Molecular
Biology
David P. Clark
_ Zn es |Table of Contents
CHAPTER 1
CHAPTER 2
‘CHAPTER 3
CHAPTER 4
CHAPTER 5
CHAPTER 6
(CHAPTER 7
‘CHAPTER 8
CHAPTER 9
‘CHAPTER 10
CHAPTER 11
CHAPTER 12
CHAPTER 13
CHAPTER 14
CHAPTER 15
CHAPTER 16
‘CHAPTER 17
CHAPTER 18
(CHAPTER 19
CHAPTER 20
CHAPTER 21
CHAPTER 22
CHAPTER 23
CHAPTER 24
‘CHAPTER 25
CHAPTER 26
Basic Geneties 1
Cells and Organisms 21
DNA, RNA and Protein 51
Genes, Genomes and DNA 75
Cell Division and DNA Replication 103
‘Transcription of Genes 132
Protein Structure and Function 154
Protein Synthesis 197
Regulation of ‘Transcription in Prokaryotes 234
Regulation of Transcription in Eukaryotes 262
Regulation at the RNA Level 281
Processing of RNA 302
Mutations 333
Recombination and Repair 368
Mobile DNA 396
Plasmids 425
Viruses 453
Bacterial Genetics 484
Diversity of Lower Eukaryotes 508
Molecular Evolution 533
Nucleic Acids: Isolation, Purification, Detection, and
Hybridization 567
Recombinant DNA Technology 599
The Polymerase Chain Reaction 634
Genomics and DNA Sequencing 662
Analysis of Gene Expression 693
Proteomics: The Global Analysis of Proteins 717
Glossary 745
Index 771Detailed Contents
CHAPTER 1 Basic Genetics
Gregor Mendel Was the Father of
Classical Gen
Genes Determine Each Step in Biochemical
Pathways
Mutants Result from Alterations in Genes
Phenotypes and Genotypes
Chromosomes Are Long, Thin Molecules
‘That Carry Genes
Different Organisms may Have Different
Numbers of Chromosomes
Dominant and Recessive Alleles
Partial Dominance, Co-Dominance,
Penetrance and Modifier Genes
Genes from Both Parents Are Mixed by
Sexual Reproduction
Sex Determination and Sex-Linked
Characteristics
Neighboring Genes Are Linked during
Inheritance
Recombination during Meiosis Ensures
Genetic Diversity
Escherichia coli Is a Model for Bacterial
Genetics
CHAPTER 2 Cells and Organisms
What Is Life?
Living Creatures Are Made of Cells
Essential Properties of a Living Cell
Prokaryotie C
ls Lack @ Nucleus
Eubacteria and Archaebacteria Are
Genetically Distinct
Bacteria Were Used for Fundamental Studies
of Cell Function
Escherichia coli (E.coli) Is a Model Bacterium
Where Are Bacteria Found in Nature?
Some Bacteria Cause Infectious Disease, but
Most Are Benefic
Eukaryotic Cells Are Sub-Divided into
Compartments,
‘The Diversity of Eukaryotes
Eukaryotes Possess Two Basic Cell Lineages
Organisms Are Classified
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Some Widely Studied Organisms Serve as
Models
Yeast Is a Widely Studied Single-Celled
Eukaryote
A Roundworm and a Fly are Model
Muhticellular Animals
Zebralish are used to Study Vertebrate
Development
Mouse and Man
Arabidopsis Serves as a Model for Plants
Haploidy, Diploidy and the Eukaryote Cell
Cycle
Viruses Are Not Living Cells
Bacterial Viruses Infect Bacteria
Human Viral Diseases Are Common,
A Variety of Subcellular Genetic Entities Exist
CHAPTER 3 DNA, RNA and
Protein
Nucleic Acid Molecules Carry Genetic
Information
Chemical Structure of Nucleic Acids
DNA and RNA Each Have Four Bases
Nucleosides Are Bases Plus Sugars;
Nucleotides Are Nucleosides Plus Phosphate
Double Stranded DNA Forms a Double H.
Base Pairs are Held Together by Hydrogen
Bonds
Complementary Strands Reveal the Secret of
Heredity
Constituents of Chromosomes
‘The Central Dogma Outlines the Flow of
Genetic Information
Ribosomes Read the Genetic Code
‘The Genetic Code Dictates the Amino Acid
Sequence of Proteins
Various Classes of RNA Have Different
Punetions
Proteins, Made of Amino Acids, Carry Out
Many Cell Funetions
‘The Structure of Proteins Has Four Levels of
Organization
Proteins Vary in Their Biological Roles
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BCHAPTER 4 Genes, Genomes and
DNA
History of DNA as the Genetic Material
How Much Genetie Information Is Necessary
to Maintain Life?
Non-Coding DNA
Coding DNA May Be Present within
Non-coding DNA
Repeated Sequences Are a Feature of DNA in
Higher Organisms
Satellite DNA Is Non-coding DNA in the Form
of Tandem Repeats
Minisatellites and VNTRs
Origin of Selfish DNA and Junk DNA
Palindromes, Inverted Repeats and Stem and
Loop Struetures,
Multiple A-Tracts Cause DNA to Bend
Supercoiling is Necessary for Packaging of
Bacterial DNA
‘Topoisomerases and DNA Gyrase
Catenated and Knotted DNA Must Be
Corrected
Local Supereoiling
Supercoiling Affect
Alternative Helical Structures of DNA Oveur
Histones Package DNA in Eukaryotes
Further Levels of DNA Packaging in
Eukaryotes
Melting Separates DNA Strands; Cooling
Anneals Them
DNA Structure
CHAPTER 5 Cell Division and
DNA Replication
Cell Division and Reproduction Are Not Always,
Identi
DNA Replication Is a Two-Stage Process
Occurring at the Replication Fork
Supercoiling Causes Problems for Replication.
‘Strand Separation Precedes DNA Synthesis
Properties of DNA Polymerase
Polymerization of Nucleotides
Supplying the Precursors for DNA Synthesis,
DNA Polymerase Blongates DNA Strands
‘The Complete Replication Fork Is Complex
iscontinuous Synthesis of DNA Requires
a Primosome
Completing the Lagging Strand
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Chromosome Replication Initiates at oriC
DNA Methylation and Attachment to the
Membrane Control Initiation of Replication
Chromosome Replication Terminates at rerC
Disentangling the Daughter Chromosomes
Cell Division in Bacteria Ovcurs alter
Replication of Chromosomes
How Long Does It Take for Bacteria to
Replicate?
The Concept of the Replicon
Replicating Linear DNA in Eukaryotes
Eukaryotic Chromosomes Have Multiple Origins
Synthesis of Eukuryotie DNA
Cell Division in Higher Organisms
CHAPTER 6 Transcription of Genes
Genes are Expressed by Making RNA
Short Segments of the Chromosome Are
“Turned into Messages
‘Terminology: Cistrons, Coding Sequences and
Open Reading Frames
How Is the Beginning of a Gene Recognized?
Manufacturing the Message
RNA Polymerase Knows Where t0 Stop
How Does the Cell Know Which Genes to
Turn On?
What Activates the Activator?
Negative Regulation Results from the Action
of Repressors
Many Regulator Proteins Bind Small Molecules
and Change Shape
‘Transcription in Eukaryotes Is More Complex:
‘Transcription of FRNA and (RNA in Bukaryotes
‘Transcription of Protein-Encoding Genes in
Eukaryotes
Upstream Elements Increase the Efficieney
of RNA Polymerase II Binding
Enhancers Control Transeription at a Distance
CHAPTER 7 Protein Structure and
Function
Proteins Are Formed from Amino Acids
Formation of Polypeptide Chains
‘Twenty Amino Acids Form Biologi
Polypeptides
Amino Acids Show Asymmetry around the
Alpha-carbon
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‘The Structure of Proteins Reflects Four Levels.
of Organization
The Secondary Structure of Proteins Relies on
Hydrogen Bonds
The Tertiary Structure of Proteins
A Variety of Forces Maintain the 3-D Structure
of Proteins
Cysteine Forms Disulfide Bonds
Multiple Folding Domains in Larger Proteins
Quaternary Structure of Proteins
Higher Level Assemblies and Self-Assembly
Cofactors and Metal Ions Are Often Associated
with Proteins
Nueleoproteins, Lipoproteins and Glycoproteins
‘Are Conjugated Proteins
Proteins Serve Numerous Cellular Functions
Protein Machines
Enzymes Catalyze Metabolic Reactions
Enzymes Have Varying Specificities
Lock and Key and Induced Fit Models Describe
Substrate Binding
Enzymes Are Named and Classified According
to the Substrate
Enzymes Act by Lowering the Energy
of Activation
‘The Rate of Enzyme Reactions
Substrate Analogs and Enzyme Inhibitors Act
at the Active Site
Enzymes May Be Directly Regulated
Allosteric Enzymes Are Affected by Signal
Molecules
Enzymes May Be Controlled by Chemical
Modification
Binding of Proteins to DNA Occurs in Several
Different Ways
Denaturation of Proteins
CHAPTER 8 Protein Synthesis
Protein Synthesis Follows a Plan
Proteins Are Gene Products
Decoding the Genetic Code
Transfer RNA Forms a Flat Cloverleaf Shape
and a Folded “L” Shape
Modified Bases Are Present in Transfer RNA
Some tRNA Molecules Read More Than
‘One Codon
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Charging the tRNA with the Amino Acid
‘The Ribosome:The Cell’s Decoding Machine
‘Three Possible Reading Frames Exist
‘The Start Codon Is Chosen
‘The Initiation Complexes Must Be Assembled
The (RNA Occupies Three Sites During
Elongation of the Polypeptide
‘Termination of Protein Synthesis Requires
Release Factors
Several Ribosomes
Message at Once
Bacterial Messenger RNA Can Code for
‘Several Proteins
Usually Read the Same
‘Transcription and Translation Are Coupled in
Bacteria
Some Ribosomes Become Stalled and Are
Rescued
Dilferences between Eukaryotic and
Prokaryotic Protein Synthesis
Initiation of Protein Synthesis in Bukaryotes
Protein Synthesis Is Halted When Resources
Are Scarce
A Signal Sequence Marks a Protein for Export
from the Cell
Molecular Chaperones Oversee Protein
Folding
Protein Synthesis Occurs in Mitochondria and
Chloroplas
Proteins Are Imported into Mitochondria and
Chloroplasts by Transtocases
Mistranslation Usually Results in Mistakes in
Protein Synthesis
The €
Unusual Amino Acids are Made in Proteins by
Post-Translational Modifications
“ode Is Not “Universal”
netic
Selenocysteine: The 21st Amino Acid
Pyrrolysine: The 22nd Amino Acid
Many Antibiotics Work by Inhibiting Protein
Synthesis
Degradation of Proteins
CHAPTER 9 Regulation of
Transcription in
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Prokaryotes
Regulation at the Level of Transcription
Involves Several StepsAlternative Sigma Faetors in Prokaryotes
Recognize Different Sets of Genes
Heat Shock Sigma Factors in Prokaryotes Are
Regulated by Temperature
Cascades of Alternative Sigma Factors Occur
in Bacillus Spore Formation
sigma Factors Inactivate Sigma;
i-sigma Factors Free Tt to Aet
Activators and Repressors Participate in
Positive and Negative Regulation
‘The Operon Model of Gene Regulation
Some Proteins May Act as Both Repressors
and Activators
Nature of the Signal Molecule
Activators and Repressors May Be Covalently
Modified
‘Two-Component Regulatory Systems
Phosphorelay Systems
Specific Versus Global Control
Crp Protein Is an Example of a Global
Control Protein
Accessory Factors and Nucleoid Binding
Proteins
Action at a Distance and DNA Looping
Anti-termination as a Control Mechanism
CHAPTER 10 Regulation of
‘Transcription in
Eukaryotes
‘Transcriptional Regulation in Eukaryotes Is
More Complex Than in Prokaryotes
Specific Transcription Factors Regulate Protein
Encoding Genes
‘The Mediator Complex Transmits Information
to RNA Polymerase
Enhancers and Insulator Sequences Segregate
DNA Functionally
Matrix Attachment Regions Allow DNA
Looping
Negative Regulation of Transeription Occurs
in Bukaryotes
Heterochromatin Causes Difficulty for Access
t DNA in Eukaryotes
Methylation of DNA in Eukaryotes Controls
Gene Expression
Silencing of Genes Is Caused by DNA
Methylation
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Genetic Imprinting in Eukaryotes Has tts
Basis in DNA Methylation Patterns
X-chromosome Inactivation Occurs in Female
XX Animals:
CHAPTER 11 Regulation at the
RNA Level
Regulation at the Level of RNA
Binding of Proteins to mRNA Controls
‘The Rate of Degradation
Some mRNA Molecules Must Be Cleaved
Before Translation
Some Regulatory Proteins May Cause
‘Translational Repression
Some Regulatory Proteins Can Activ:
‘Translation
‘Translation May Be Regulated by
Antisense RNA
Regulation of Translation by Alterations to
the Ribosome
RNA Interference (RNAi)
Amplification and Spread of RNAi
Experimental Administration of SRNA
PTGS in Plants and Quelling in Fungi
Micro RNA—A Class of Small
Regulatory RNA.
Premature Termination Causes Attenuation of
RNA Transcription
Riboswitches—RNA Acting Directly as a
Control Mechanism
CHAPTER 12 Processing of RNA
RNA js Processed in Several Ways
Coding and Noa-Coding RNA.
Processing of Ribosomal and Transfer RNA
Eukaryotic Messenger RNA Contains a Cap
and Tail
Capping is the First Step in Maturation of
mRNA,
A Poly(A) Tail is Added to Eukaryotic mRNA
Introns are Removed from RNA by Splicing
Different Classi
Splicing Me
Aliernative Splicing Produces Multiple Forms of
RNA
of Intron Show Different
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Inteins and Protein Splicing
Modification of rRNA Requires Guide
RNA Editing Involves Altering the Base
Sequence
Transport of RNA out of the Nucleus
Degradation of mRNA
Nonsense Mediated Decay of mRNA
CHAPTER 13 Mutations
Mutations Alter the DNA Sequence
‘The Major Types of Mutation
Base Substitution Mutations
Missense Mutations May Have Major or
Minor Eficets
Nonsense Mutations Cause Premature
Polypeptide Chain Termination
Deletion Mutations Result in Shortened or
Absent Proteins
Insertion Mutations Commonly Disrupt
Existing Genes
Frameshift Mutations Sometimes Produce
Abnormal Proteins.
DNA Rearrangements Include Inversions,
1 Duplications
Phase Variation Is Due to Reversible DNA
Alterations
Translocation:
Silent Mutations Do Not Alter the Phenotype
Chemical Mutagens Damage DNA
Radiation Causes Mutations
Spontaneous Mutations Can Be C
DNA Polymerase Errors
Mutations Can Result from Mispairing and
Recombination
Spontaneous Mutation Can Be the Result of
Tautomerization
used by
Spontancous Mutation Can Be Caused by
Inherent Chemical Instability
Mutations Occur More Frequently at Hot Spots
How Often Do Mutations Occur?
Reversions Are Genetic Alterations That
Change the Phenotype Back to Wild-type
Reversion Can Occur by Compensatory
Changes in Other Genes
Altered Decoding by Transfer RNA May
Cause Suppression
Mutagenic Chemicals Can Be Detected by
Reversion
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Experimental Isolation of Mutations
In Vivo versus In Vitro Mi
Site-Directed Mutagenesis
CHAPTER 14 Recombination and
Repair
Overview of Recombination
Molecular Basis of Homologous Recombination
Single-Strand Invasion and Chi Sites
Site-Specific Recombination
Recombination in Higher Organisms
Overview of DNA Repair
DNA Mismatch Repair §
General Excision Repair Sy
DNA Repair by Excision of Specific Bases
Specialized DNA Repair Mechanisms
Photoreactivation Cleaves Thymine Dimers
‘Transcriptional Coupling of Repair
Repair by Recombination
SOS Error Prone Repair in Bacteria
Repair
Double-Strand Repair in Eukaryotes
Gene Conversion
tem
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in Bukaryotes
CHAPTER 15 Mobile DNA
Sub-Cellular G
Creatures
Most Mobile DNA Consists of Transposable
Elements
ments as Gene
netic
‘The Essential Parts of a Transposon
Insertion Sequences—the Simplest Transposons.
Movement by Conservative Transposi
Complex Transposons Move by Replicative
‘Transposition
Replicative and Conservative Transposition are
Related
Composite Transposons
‘Transposition may Rearrange Host DNA
‘Transposons in Higher Life Forms
Retro-Elements Make an RNA Copy
Repetitive DNA of Mamm:
Retro-Insertion of Host-Derived DNA.
Retrons Encode Bacterial Reverse Transcriptase
The Multitude of Transposable Elements
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a7Bacteriophage Mu is a Transposon
Conjugative Transposons
Integrons Collect Genes for Transposons
Junk DNA and Selfish DNA
‘Homing Introns
CHAPTER 16 Plasmids
Plasmids as Replicons
General Properties of Plasmids
Plasmid Families and Incompatibility
Occasional Plasmids are Linear or Made of
RNA
Plasmid DNA Replicates by Two Alternative
Methods
Control of Copy Number by Antisense RNA
Plasmid Addiction and Host Killing Functions
Many Plasmids Help their Host Cells
Antibiotic Res
Mechanism of Antibiotic Resistance
Resistance to Beta-Lactam Antibiotics
Resistance to Chloramphenicol
Resistance to Aminogly
Resistance to Tetracycline
Resistance to Sulfonamides and Trimethoprim
Plasmids may Provide Aggee
Most Colicins Kill by One of Two Different
Mechanisms
Bacteria are Immune to their own Colicins
tance Plasmids
vosides
ve Characters
Colicin Synthesis and Release
Virulence Plasmids
‘Ti-Plasmids are Transferred from Bacteria to
Plants
The 2 Micron Plasmid of Yeast
Certain DNA Molecules may Behave as
‘Viruses ot Plasmids
CHAPTER 17 Viruses
Viruses are Infectious Packages of Genetic
Information
Life
Bacterial Viruses are Known as Bacteriophage
Lysogeny or Lateney by Integration
‘The Great Diversity of Viruses
Small Single-Stranded DNA Viruses of Bacteria
Complex Bacterial Viruses with Double
‘Stranded DNA,
cle of a Virus
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DNA Viruses of Higher Organisms
Viruses with RNA Genomes Have Very Few
Genes
Bacterial RNA Viruses
Double Stranded RNA Viruses of Animals
Positive-Stranded RNA Viruses Make
Polyproteins
Strategy of Negative-Strand RNA Viruses
Plant RNA Viruses
Retroviruses Use both RNA and DNA
Genome of the Retrovirus
Subviral Infectious Agents
Satellite Viruses
Viroids are Naked Molecules of Infectious RNA
Prions are Infectious Proteins
CHAPTER 18 Bacterial Genetics
Reproduction versus Gene Transfer
Fate of the Incoming DNA after Uptake
is Gene Transfer by Naked DNA
Transformation as Proof that DNA is the
Genetic Material
‘Transformation in Nature
Gene Transfer by Virus—Transduction
Generalized Transduction
Specialized Transduction
‘Transfer of Plasmids between Bacteria
‘Transfer of Chromosomal Genes Requires
Plasmid Integration
Gene Transfer among Gram-Positive Bacteria
‘Transform:
Archacbacterial Genetics
Whole Genome Sequencing
CHAPTER 19 Diversity of
Lower Eukaryotes
Origin of the Eukaryotes by Symbiosis
The Genomes of Mitochondria and Chloroplasts,
Primary and Secondary Endosymbiosis
Is Malaria Really a Plant?
Symbiosis: Parasitism versus Mutualism
Bacerial Endosymbionts of Killer Paramecium
s Buchnera an Organelle or a Bacterium’?
Ciliates have Two Types of Nucleus
‘Trypanosomes Vary Surface Proteins to Outwit
the Immune System
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