Genetics and Molecular Biology

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Genetics and Molecular Biology

Genetics and Molecular Biology



An Overview of Cell Structure and Function


Cellís Need for Immense Amounts of Information
Rudiments of Prokaryotic Cell Structure
Rudiments of Eukaryotic Cell Structure
Packing DNA into Cells
Moving Molecules into or out of Cells
Diffusion within the Small Volume of a Cell
Exponentially Growing Populations
Composition Change in Growing Cells
Age Distribution in Populations of Growing Cell

Nucleic Acid and Chromosome Structure


The Regular Backbone of DNA
Grooves in DNA and Helical Forms of DNA
Dissociation and Reassociation of Base-paired Strands
Reading Sequence Without Dissociating Strands
Electrophoretic Fragment Separation
Bent DNA Sequences
Measurement of Helical Pitch
Topological Considerations in DNA Structure
Generating DNA with Superhelical Turns
Measuring Superhelical Turns
Determining Lk, Tw, and Wr in Hypothetical Structures
Altering Linking Number
Biological Significance of Superhelical Turns
The Linking Number Paradox of Nucleosomes
General Chromosome Structure
Southern Transfers to Locate Nucleosomes on Genes
ARS Elements, Centromeres, and Telomeres

DNA Synthesis


DNA Synthesis
Proofreading, Okazaki Fragments, and DNA Ligase - Enzymology
Detection and Basic Properties of DNA Polymerases - Enzymology
In vitro DNA Replication - Enzymology
Error and Damage Correction - Enzymology
DNA Replication Areas In Chromosomes - Physiological Aspects
Bidirectional Replication from E. coli Origins - Physiological Aspects
The DNA Elongation Rate - Physiological Aspects
Constancy of the E. coli DNA Elongation Rate - Physiological Aspects
Regulating Initiations - Physiological Aspects
Gel Electrophoresis Assay of Eukaryotic Replication Origins - Physiological Aspects
How Fast Could DNA Be Replicated? - Physiological Aspects

RNA Polymerase and RNA Initiation


Measuring the Activity of RNA Polymerase
Concentration of Free RNA Polymerase in Cells
The RNA Polymerase in Escherichia coli
Three RNA Polymerases in Eukaryotic Cells
Multiple but Related Subunits in Polymerases
Multiple Sigma Subunits
Structure of Promoters
Enhancers
Enhancer-Binding Proteins
DNA Looping in Regulating Promoter Activities
Steps of the Initiation Process
Measurement of Binding and Initiation Rates
Relating Abortive Initiations to Binding and Initiating
Roles of Auxiliary Transcription Factors
Melted DNA Under RNA Polymerase

TranscriptionTermination and RNA Processing


Polymerase Elongation Rate
Transcription Termination at Specific Sites
RNA Termination
Processing Prokaryotic RNAs After Synthesis
S1 Mapping to Locate 5í and 3í Ends of Transcripts
Caps, Splices, Edits, and Poly-A Tails on Eukaryotic RNAs
The Discovery and Assay of RNA Splicing
Involvement of the U1 snRNP Particle in Splicing
Splicing Reactions and Complexes
The Discovery of Self-Splicing RNAs
A Common Mechanism for Splicing Reactions
Other RNA Processing Reactions

Protein Structure


Protein Structure
The Amino Acids - Protein Structure
Peptide Bond - Protein Structure
Electrostatic Forces that Determine Protein Structure - Protein Structure
Hydrogen Bonds and the Chelate Effect - Protein Structure
Hydrophobic Forces - Protein Structure
Thermodynamic Considerations of Protein Structure
Structures within Proteins
Alpha Helix, Beta Sheet, and Beta Turn - Protein Structure
Calculation of Protein Tertiary Structure
Secondary Structure Predictions - Protein Structure
Structures of DNA-Binding Proteins
Salt Effects on Protein-DNA Interactions
Locating Specific Residue-Base Interactions - Protein Structure

Protein Synthesis


Activation of Amino Acids During Protein Synthesis
Fidelity of Aminoacylation - Protein Synthesis
How Synthetases Identify the Correct tRNA Molecule
Decoding the Message - Protein Synthesis
Base Pairing between Ribosomal RNA and Messenger
Experimental Support for the Shine-Dalgarno Hypothesis - Protein Synthesis
Eukaryotic Translation and the First AUG - Protein Synthesis
Tricking the Translation Machinery into Initiating - Protein Synthesis
Protein Elongation - Protein Synthesis
Peptide Bond Formation - Protein Synthesis
Translocation - Protein Synthesis
Termination, Nonsense, and Suppression - Protein Synthesis
Chaperones and Catalyzed Protein Folding - Protein Synthesis
Resolution of a Paradox - Protein Synthesis
Messenger Instability - Protein Synthesis
Protein Elongation Rates - Protein Synthesis
Directing Proteins to Specific Cellular Sites - Protein Synthesis
Verifying the Signal Peptide Model - Protein Synthesis
The Signal Recognition Particle and Translocation - Protein Synthesis
Expectations for Ribosome Regulation - Protein Synthesis
Proportionality of Ribosome Levels and Growth Rates - Protein Synthesis
Regulation of Ribosome Synthesis - Protein Synthesis
Balancing Synthesis of Ribosomal Components - Protein Synthesis

Genetics


Genetics Mutations
Point Mutations, Deletions, Insertions, and Damage
Classical Genetics of Chromosomes
Complementation, Cis, Trans, Dominant, and Recessive
Mechanism of a trans Dominant Negative Mutation
Genetic Recombination
Genetics Mapping by Recombination Frequencies
Genetics: Mapping by Deletions
Heteroduplexes and Genetic Recombination
Genetics: Branch Migration and Isomerization
Elements of Recombination in E. coli, RecA, RecBCD, and Chi
Genetic Systems
Growing Cells for Genetics Experiments
Testing Purified Cultures, Scoring
Isolating Auxotrophs, Use of Mutagens and Replica Plating
Genetic Selections
Mapping with Generalized Transducing Phage
Principles of Bacterial Sex
Elements of Yeast Genetics
Elements of Drosophila Genetics
Isolating Mutations in Muscle or Nerve in Drosophila
Fate Mapping and Study of Tissue-Specific Gene Expression

Genetic Engineering and Recombinant DNA


Isolation of DNA
Biology of Restriction Enzymes
Cutting DNA with Restriction Enzymes
Isolation of DNA Fragments
Joining DNA Fragments
Vectors: Selection and Autonomous DNA Replication
Plasmid Vectors
A Phage Vector for Bacteria
Vectors for Higher Cells
Putting DNA Back into Cells
Cloning from RNA
Plaque and Colony Hybridization for Clone Identification
Walking Along a Chromosome to Clone a Gene
Arrest of Translation to Assay for DNA of a Gene
Chemical DNA Sequencing
Enzymatic DNA Sequencing

Advanced Genetic Engineering


Finding Clones from a Known Amino Acid Sequence
Finding Clones Using Antibodies Against a Protein
Southern, Northern, and Western Transfers - Genetic Engineering
Polymerase Chain Reaction
Isolation of Rare Sequences Utilizing PCR
Physical and Genetic Maps of Chromosomes
Chromosome Mapping
DNA Fingerprinting - Forensics
Megabase Sequencing
Footprinting, Premodification and Missing Contact Probing
Antisense RNA: Selective Gene Inactivation
Hypersynthesis of Proteins
Altering Cloned DNA by in vitro Mutagenesis
Mutagenesis with Chemically Synthesized DNA

Repression and the lac Operon


Repression and the lac Operon
Role of Inducer Analogs in the Study of the lac Operon
Proving lac Repressor is a Protein
An Assay for lac Repressor
Difficulty of Detecting Wild Type lac Repressor
Detection and Purification of lac Repressor
Repressor Binds to DNA: The Operator is DNA
Migration Retardation Assay and DNA Looping - Repression and the lac Operon
Isolation and Structure of Operator - Repression and the lac Operon
In vivo Affinity of Repressor for Operator
DNA-binding Domain of lac Repressor
A Mechanism for Induction - Repression and the lac Operon

Induction Repression and the araBAD Operon


Sugar Arabinose and Arabinose Metabolism
Genetics of the Arabinose System
Detection and Isolation of AraC Protein
Repression by AraC
Regulating AraC Synthesis
Binding Sites of the ara Regulatory Proteins
DNA Looping and Repression of araBAD
In vivo Footprinting Demonstration of Looping
How AraC Protein Loops and Unloops
Why Looping is Biologically Sensible
Why Positive Regulators are a Good Idea

Attenuation and the trp Operon


Attenuation and the trp Operon
Rapid Induction Capabilities of the trp Operon
Serendipitous Discovery of trp Enzyme Hypersynthesis
Early Explorations of the Hypersynthesis
trp Multiple Secondary Structures in trp Leader RNA
Coupling Translation to Termination
RNA Secondary Structure and the Attenuation Mechanism
Other Attenuated Systems: Operons, Bacillus subtilis and HIV

Lambda Phage Genes and Regulatory Circuitry


Physical Structure of Lambda
Genetic Structure of Lambda
Lysogeny and Immunity
Lambdaís Relatives and Lambda Hybrids
Lambda Adsorption to Cells
Early Transcription of Genes N and Cro
N Protein and Antitermination of Early Gene Transcription
Role of Cro Protein
Initiating DNA Synthesis with the O and P Proteins
Proteins Kil, ?, ?, and Exo
Q Protein and Late Protein Synthesis
Lysis
Chronology of Becoming a Lysogen
Site for Cro Repression and CI Activation
Cooperativity in Repressor Binding and its Measurement
Need for and the Realization of Hair-Trigger Induction
Induction from the Lysogenic State
Entropy, a Basis for Lambda Repressor Inactivation

Xenopus 5S RNA Synthesis


Biology of 5S RNA Synthesis in Xenopus
In vitro 5S RNA Synthesis
TFIIIA Binding to the Middle of its Gene as Well as to RNA
Switching from Oocyte to Somatic 5S Synthesis
Structure and Function of TFIIIA

Regulation of Mating Type in Yeast


Regulation of Mating Type in Yeast
Yeast Cell Cycle
Mating Type Conversion in Saccharomyces cerevisiae
Cloning the Mating Type Loci in Yeast
Transfer of Mating Type Gene Copies to an Expression Site
Structure of the Mating Type Loci
Expression and Recombination Paradoxes
Silencing HML and HMR
Isolation of ?2 Protein
?2 and MCM1
Sterile Mutants, Membrane Receptors and G Factors
DNA Cleavage at the MAT Locus
DNA Strand Inheritance and Switching in Fission Yeast

Genes Regulating Development


General Considerations on Signaling
Outline of Early Drosophila Development
Classical Embryology
Using Genetics to Begin Study of Developmental Systems
Cloning Developmental Genes
Enhancer Traps for Detecting and Cloning Developmental Genes
Expression Patterns of Developmental Genes
Similarities Among Developmental Genes
Overall Model of Drosophila Early Development

Lambda Phage Integration and Excision


Mapping Integrated Lambda
Simultaneous Deletion of Chromosomal and Lambda DNA
DNA Heteroduplexes Prove that Lambda Integrates
Gene Order Permutation and the Campbell Model
Isolation of Integration-Defective Mutants
Isolation of Excision-Deficient Mutants
Properties of the int and xis Gene Products
Incorrect Excision and gal and bio Transducing Phage
Transducing Phage Carrying Genes Other than gal and bio
Use of Transducing Phage to Study Integration and Excision
The Double att Phage, att.squ
Demonstrating Xis is Unstable
Inhibition By a Downstream Element
In vitro Assay of Integration and Excision
Host Proteins Involved in Integration and Excision
Structure of the att Regions
Structure of the Intasome
Holliday Structures and Branch Migration in Integration

Transposable Genetic Elements


Transposable Genetic Elements
IS Elements in Bacteria
Structure and Properties of IS Elements
Discovery of Tn Elements
Structure and Properties of Tn Elements
Inverting DNA Segments by Recombination, Flagellin Synthesis
Mu Phage As a Giant Transposable Element
An Invertible Segment of Mu Phage
In vitro Transposition, Threading or Global Topology?
Hopping by Tn10
Retrotransposons in Higher Cells
An RNA Transposition Intermediate
P Elements and Transformation
P Element Hopping by Chromosome Rescue

Generating Genetic Diversity


Generating Genetic Diversity: Antibodies
Basic Adaptive Immune Response
Telling the Difference Between Foreign and Self
Number of Different Antibodies Produced
Myelomas and Monoclonal Antibodies
Structure of Antibodies
Many Copies of V Genes and Only a Few C Genes
J Regions - Generating Genetic Diversity: Antibodies
D Regions in H Chains
Induced Mutations and Antibody Diversity
Class Switching of Heavy Chains
Enhancers and Expression of Immunoglobulin Genes
AIDS Virus
Engineering Antibody Synthesis in Bacteria
Assaying for Sequence Requirements of Gene Rearrangements
Cloning the Recombinase

Biological Assembly Ribosomes and Lambda Phage


Biological Assembly, Ribosomes and Lambda Phage
RNAse and Ribosomes
Global Structure of Ribosomes
Assembly of Ribosomes
Experiments with in vitro Ribosome Assembly
Determining Details of Local Ribosomal Structure
Lambda Phage Assembly: General Aspects
Geometry of Capsids
Structure of the Lambda Particle
Head Assembly Sequence and Host Proteins
Packaging the DNA and Formation of the cos Ends
Formation of the lambda Tail
In vitro Packaging

Chemotaxis


Chemotaxis
Assaying Chemotaxis
Fundamental Properties of Chemotaxis
Genetics of Motility and Chemotaxis
How Cells Swim
Mechanism of Chemotaxis
Energy for Chemotaxis
Adaptation
Methylation and Adaptation
Phosphorylation and the Rapid Response

Oncogenesis Molecular Aspects


Bacterially Induced Tumors in Plants
Transformation and Oncogenesis by Damaging the Chromosome
Identifying a Nucleotide Change Causing Cancer
Retroviruses and Cancer
Cellular Counterparts of Retroviral Oncogenes
Identification of the src and sis Gene Products
DNA Tumor Viruses
Recessive Oncogenic Mutations, Tumor Suppressors
ras-fos-jun Pathway
Directions for Future Research in Molecular Biology



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