Bacterial, Phage and Molecular Genetics: An Experimental Course

ISBN-13: 9783540076025 / Angielski / Miękka / 1976 / 242 str.

U. Winkler; W. Ruger; W. Wackernagel

Opis

During the mid-forties bacteria and phages were dis- covered to be suitable objects for the study of genet- ics. Genetic phenomena such as mutation and recombina- tion, which had already been known in eukaryotes for a long time, were now shown to exist in bacteria and phages as well. New phenomena as lysogeny and trans- duction were discovered, which gained great importance beyond the field of microbial genetics. Bacteria and phages are of small size, multiply rapid- ly, and have chemically defined growth requirements. Many selective procedures can be applied to screen for rarely occurring mutants or recombinants. Therefore, they offered ideal conditions to investigate genetic processes and to interpret them in molecular terms. Many new methods were developed (e. g. CsCl density gradient centrifugation) and old techniques were im- proved and modified for new purposes (e. g. chemical mutagenesis). Hypotheses, such as the semiconservative replication of DNA, mutation by transition and trans- version and operon regulation, have had an extraordi- narily stimulating effect on the research in general genetics. Thus, in the past two decades, from the ge- netics of microbes (including fungi) the field of mo- lecular genetics developed. Many text books compete in presenting the latest knowledge on this subject. But to date there are only a few laboratory manuals which introduce the student of biology to the manifold ex- perimental techniques of microbial and molecular gene- tics. This laboratory manual is an attempt to redress the balance.

Spis treści

I. General.- A. Abbreviations and Expressions Frequently Used.- B. Basic Equipment for the Experiments.- C. Calculation of Titers and Some Statistical Methods.- D. Technical Literature.- II. Experiments and Problems.- A. Phage Growth and Ultracentrifugation.- 1. Large Scale Production of Phage T4.- 2. Purification and Concentration of Phage T4.- a) Dextransulfate — PEG Two-Phase Technique.- 3. Purification and Concentration of Phage T4.- b) Centrifugation with and without CsCl.- 4. Determination of the Density of Phages by CsCl Gradient Centrifugation.- 5. Estimation of Sedimentation Constants by Sucrose Gradient Centrifugation 43 Problems.- B. Nucleic Acids and Transcription.- 6. Isolation of DNA from T4 or T5 Phages.- 7. Thermal De- and Renaturation of DNA from Different Organisms.- 8. Isolation of 3H-Uridine-labeled mRNA from Bacteria Infected with T4.- 9. Hybridization of Phage-DNA and Phage-mRNA on Nitrocellulose Filters.- 10. Time of Gene Transcription – Determined by Phenocopying with 5-Fluorouracil 72 Problems.- C. Mutation and Photobiology.- 11 Genotype and Phenotype.- 12. Proof of the Random Distribution of Spontaneous Mutations.- 13. Mutagenic and Inactivating Action of Ultraviolet Light on Phage Kappa.- 14. Reversible Photochemical Alteration of Cytidylic Acid.- 15. Chemical Induction of Forward and Back Mutations in Bacteria.- Problems.- D. Transfer and Recombination of Genetic Material.- 16. Conjugation of E. coli Bacteria.- 17. Two- and Three-Factor Crosses with Phage Kappa.- 18. Transduction of Linked and Unlinked Genetic Markers with Phage P1.- 19. Transfer and Elimination of a Plasmid (F’lac).- 20. Induction of Lysogenic and Bacterio-cinogenic Bacteria.- 21. Transfection of E. coli Spheroplasts with DNA of Phage Lambda.- Problems.- E. Phenotypic Expression.- 22. Determination of a Metabolic Block by Auxanography and Syntrophy.- 23. Complementation of Nonsense Mutants of Phage Kappa 1.- 24. Constitutive and Induced Synthesis of ß-Galactosidase.- 25. The Morphopoesis of T4 Phages.- Problems.- III. Appendix.- A. Nutrient Media and Solutions.- B. Strains of Bacteria and Phages.- C. Sources of Supplies for Experiments.- D. The Recording of Scientific Experiments..- IV. Experimental Results and Answers to the Problems.- A. Experimental Results.- B. Answers to the Problems.- The Genetic Code.