Single Cell Bacteria
Blackwell Publishing

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.