Advanced Molecular Genetics

Abstract

1 Basic Methods.- 1.1 Genetic Transfer in Prokaryotes: Transformation, Transduction, and Conjugation.- 1.2 Isolation of Plasmid DNA.- 1.3 Characterization of Plasmid DNA by Agarose Gel Electrophoresis.- 1.4 Characterization of Plasmid in Wild Strains of Bacteria.- 1.5 Mobilization of Host Chromosomal Genes and Vector Plasmids in E. coli by the P-Type Plasmid R68.45.- 1.6 Characterization of Plasmid Relaxation Complexes.- 1.7 Use of the BAL31 Exonuclease to Map Restriction Endonucleases Cleavage Sites in Circular Genomes.- 1.8 IncPl Typing of Plasmid DNA by Southern Hybridization.- 1.9 Molecular Epidemiology by Colony Hybridization Using Cloned Genes.- 2 Mutagenesis.- 2.1 Isolation of Suppressible Mutations by Hydroxylamine Mutagenesis in Vitro.- 2.2 Transposition of Tni to the Phage PI Genome: Isolation of Restriction Deficient Mutants.- 2.3 In Vivo Genetic Engineering: Use of Transposable Elements in Plasmid Manipulation and Mutagenesis of Bacteria Other Than E. coli.- 2.4 Generation of Deletion Mutations in Vitro with the BAL31 Exonuclease.- 3 Gene Cloning.- 3.1 Gene Cloning: An Introduction.- 3.2 Ligation of Cohesive-Ended and Flush-Ended DNA Fragments.- 3.3 Cloning with Plasmid Vectors.- 3.4 Mini-Plasmid Formation: The Cloning of Replication Origins.- 3.5 Gene Cloning with Bacteriophage ?.- 3.6 Cloning with Cosmids.- 4 Gene Expression.- 4.1 Synthesis of Plasmid-Encoded Polypeptides in Maxicells.- 4.2 Synthesis of Bacteriophage and Plasmid-Eneoded Polypeptides in Minicells.- 4.3 Determination of Coding Regions on Multicopy Plasmids: Analysis of the Chloramphenicol Acetyltransferase Gene of Plasmid pACYC184.- 4.4 Identification of Gene Products by Coupled Transcription- Translation of DNA Fragments in Cell-Free Extracts of E. coli.- 5 DNA Sequencing.- 6 Electron Microscopy.- 7 Transcription.- 7.1 Mapping of RNA Polymerase Binding Sites by Electron Microscopy.- 7.2 Determination of the Startpoints and Orientation of in Vitro Transcripts by Electron Microscopy of R-Loops.- 8 DNA Replication.- 8.1 Localization of Origins of Plasmid DNA Replication.- 8.2 Plasmid DNA Replication in Vitro.