Fundamental Bacterial Genetics

1. Introduction To The Cell.

The Molecules That Make Up A Cell.

The Bacterial Cell: A Quick Overview.

How Do Cells Grow?.

What Is Genetics?.

Summary.

2. The Bacterial Dna Molecule.

The Structure Of DNA And RNA.

Deoxyribonucleosides And Deoxyribonucleotides.

DNA Is Only Polymerized 5’ To 3’.

Double-Stranded Dna.

Supercoiling Double-Stranded Dna.

Replication Of The Escherichia Coli Chromosome.

Constraints That Influence Dna Replication.

The Replication Machinery.

Dna Polymerases.

Dnag Primase.

Replication Of Both Strands.

Theta Mode Replication.

Minimizing Mistakes In Dna Replication.

The Dna Replication Machinery As Molecular Tools.

Summary.

3. Mutations.

Phenotype And Genotype.

Classes Of Mutations.

Point Mutations And Their Consequences.

Measuring Mutations: Rate And Frequency.

Spontaneous And Induced Mutations.

Errors During Dna Replication: Incorporation Errors.

Errors Due To Tautomerism.

Spontaneous Alteration By Depurination.

Spontaneous Alteration By Deamination.

Alterations By Spontaneous Genetic Rearrangement.

Alterations Caused By Transposition.

Induced Mutations.

Chemicals That Mimic Normal Dna Bases: Base Analogues.

Chemicals That React With Dna Bases: Base Modifiers.

Chemicals That Bind Dna Bases: Intercalators.

Mutagens That Physically Damage The Dna: Ultraviolet Light And Ionizing.

Radiation.

Mutator Strains.

Reverting Mutations.

Suppression.

Ames Test.

How Have We Exploited Bacterial Mutants.

Summary.

4. Dna Repair.

Lesions That Constitute Dna Damage.

Reverse, Excise Or Tolerate?.

Mechanisms That Reverse Dna Damage.

Photoreactivation.

O6-Methylguanine Or O4-Methylthymine Methyltransferase.

Mechanisms That Excise Dna Damage.

Uvrabc Directed Nucleotide Excision Repair.

Muthls Methyl Directed Mismatch Repair.

Very Short Patch Repair.

Glycosylases.

Uracil-N-Glycosylase Coupled With Ap Excision Repair.

Deaminated Bases Removed By Dna Glycosylase.

Alkylated Bases Removed By Dna Glycosylase.

Mutm/Muty: Oxidative Damage.

N-Glycosylases Specific For Pyrimidine Dimmers.

Mechanisms That Tolerate Dna Damage.

Transdimer Synthesis.

Post Replication/Recombinational Repair (Prr).

Introduction To The Sos Regulon.

Summary.

5. Recombination:.

Homologous Recombination.

Models For Homologous Recombination.

The Holliday Or Double-Strand Invasion Model Of Recombination.

An Alternative To The Holliday Model: The Single Strand Invasion Model Of Meselson And Radding.

Further Enzymatic Considerations.

Site-Specific Recombination.

A Typical Site-Specific Recombinational Event.

Bacteriophage l:A Model For Site-Specific Recombination.

Other Examples Of Site-Specific Recombination.

Illegitimate Recombination.

Summary.

6. Transposition.

The Structure Of Transposons.

The Frequency Of Transposition.

The Two Types Of Transposition Reactions.

The Transposition Machinery.

The Transposition Machinery; Accessory Proteins Encoded By The Transposon.

The Transposition Machinery: Accessory Proteins Encoded By The Host.

Non-Replicative Transposition.

Replicative Transposition.

Does The Formation Of A Cointegrate Predict The Transposition Mechanism?.

The Fate Of The Donor Site.

Target Immunity.

Transposons As Molecular Tools.

Summary.

7. Bacteriophage.

The Structure Of Phage.

The Life Cycle Of A Bacteriophage.

Lytic-Lysogenic Options.

The l Lifecycle.

l Adsorption.

l Dna Injection.

Protecting The l Genome In The Bacterial Cytoplasm.

What Happens To The l Genome After It Is Stabilized?.

l And The Lytic-Lysogenic Decision.

The l Lysogenic Pathway.

The l Lytic Pathway.

Dna Replication During The l Lytic Pathway.

Making l Phage.

Getting Out Of The Cell-The l S And R Proteins.

Induction Of By The Sos System.

Superinfection.

Restriction And Modification Of Dna.

The Lifecycle Of M13-M13 Adsorption And Injection.

Protection Of The M13 Genome.

M13 Dna Replication.

M13 Phage Production And Release From The Cell.

The Lifecycle Of P1.

Adsorption, Injection And Protection Of The Genome.

P1 Dna Replication And Phage Assembly.

The Location Of The P1 Prophage In A Lysogen.

P1 Transducing Particles.

The Lifecycle Of T4-T4 Adsorption And Injection.

T4rii Mutations And The Nature Of The Genetic Code.

Summary.

8. Transduction.

Generalized Transduction Vs Specialized Transduction.

P1 As A Model For Generalized Transducing Phage.

Packaging The Chromosome.

Moving Pieces Of The Chromosome From One Cell To Another.

Identifying Transduced Bacteria: Selection Vs Screen.

Carrying Out A Transduction.

Uses For Transduciton.

Two Factor Crosses To Determine Gene Linkage.

Mapping The Order Of Genes- Three Factor Crosses.

Uses For Transduction-Strain Construction.

Uses For Transduction-Localized Mutagenesis.

Specialized Transducing Phage.

Making Merodiploids With Specialized Transducing Phage.

Moving Mutations From Plasmids To Specialized Transducing Phage To The.

Chromosome.

Summary.

9. Natural Plasmids.

Origins Of Replication.

Plasmid Copy Number.

Setting The Copy Number.

Plasmid Incompatibility.

Plasmid Amplification.

Other Genes That Can Be Carried By Plasmids.

Plasmids Can Be Circular Or Linear Dna.

Broad Host Range Plasmids.

Moving Plasmids From Cell To Cell.

Summary.

10. Conjugation:.

The F Factor.

The R Factors.

The Conjugation Machinery.

Transfer Of The Dna.

Surface Exclusion.

F, Hfr Or F-Prime.

Formation Of The Hfr.

Transfer Of Dna From An Hfr To Another Cell.

Formation Of F-Primes.

Transfer Of F-Primes From One Cell To Another.

Genetic Uses Of F-Primes.

Genetic Uses Of Hfr Strains-Mapping Genes On The E. Coli Chromosome Using Hfr.

Crosses.

The 50% Rule.

Using Several Hfr Strains To Cover The Chromosome.

Mobilization Of Non-Conjugatible Plasmids By R And F.

Conjugation From Prokaryotes To Eukaryotes.

Summary.

11. Transformation:.

Natural Competency.

The Process Of Natural Transformation.

The Machinery Of Naturally Transformable Cells.

Artificial Transformation.

Transformation As A Genetic Tool: Gene Mapping.

Transformation As A Molecular Tool.

Summary.

12. Gene Expression And Regulation.

The Players In The Regulation Game.

Operons And Regulons.

Repression Of The Lac Operon.

Activation Of The Lac Operon By Cyclic Amp And The Cap Protein.

Regulation Of The Tryptophan Biosynthesis Operon By Attenuation.

Regulation Of The Heat Shock Regulon By An Alternative Sigma Factor, Mrna Stability And Proteolysis.

Regulation Of The Sos Regulon By Proteolytic Cleavage Of The Repressor.

Two Component Regulatory Systems, Signal Transduction And The Cps Regulon.

Summary.

13. Plasmids, Bacteriophage And Transposons As Tools.

What Is A Cloning Vector?.

Why Not Use Naturally Occurring Plasmids As Vectors?.

The Importance Of Copy Number.

An Example Of How A Cloning Vector Works-Pbr322.

Multiple Cloning Sites.

Determining Which Plasmids Contain An Insert.

Expression Vectors.

Vectors For Purifying The Cloned Gene Product.

Vectors For Localizing The Gene Product.

Vectors For Studying Gene Expression.

Shuttle Vectors.

Artificial Chromosomes.

Constructing Phage Vectors.

Suicide Vectors.

Phage Display Vectors.

Combining Phage Vectors And Transposons.

Summary.

14. DNA Cloning:.

Isolating DNA From Cells - Plasmid DNA Isolation.

Isolating DNA From Cells - Chromosomal DNA Isolation.

Cutting DNA Molecules.

Type I Restriction-Modification Systems.

Type II Restriction-Modification Systems.

Type III Restriction-Modification Systems.

Restriction-Modification As A Molecular Tool.

Generate Double Stranded Breaks In DNA By Shearing The Dna.

Joining DNA Molecules.

Manipulating The Ends Of Molecules.

Visualizing The Cloning Process.

Constructing Libraries Of Clones.

DNA Detection – Southern Blotting.

DNA Amplification: Polymerase Chain Reaction.

Adding Novel Dna Sequences To The Ends Of A Pcr Amplified Sequence.

Site Directed Mutagenesis Using Pcr.

Cloning And Expressing A Gene.

Dna Sequencing Using Dideoxy Sequencing.

Dna Sequence Searches.

Summary.

15. Bioinformatics And Proteomics.

Bioinformatics.

Strategies For Sequencing Genomes.

Bacterial Genomes.

Analyzing Genomes.

The E. Coli K-12 Genome.

Proteomics.

Techniques For Examining The Proteome-Sds-Page And 2-Dimensional Sds-Page.

Techniques For Examining The Proteome-Microarray Technology.

Summary.

Glossary.

Additional References.