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Classical Bacterial Genetics

Classical Bacterial Genetics. A. Conjugation B. Conditional lethal mutations C. Discovery of lac regulatory genes D. Suppressors & Protein Targeting. Conditional Lethal Mutations. What kind of genes whose mutation is lethal? How could one get mutants of such kind of genes?

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Classical Bacterial Genetics

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  1. Classical Bacterial Genetics A. Conjugation B. Conditional lethal mutations C. Discovery of lac regulatory genes D. Suppressors & Protein Targeting

  2. Conditional Lethal Mutations • What kind of genes whose mutation is lethal? • How could one get mutants of such kind of genes? • What features of such kind of mutations have in common?

  3. DNA polymerase I 1. What is the physiological role of DNA polymerase I in vivo? -- Is it essential for growth? -- Is it involved in DNA repair? In DNA recombination? 2. What enzymatic activities does DNA polymerase I possess? -- What is the physiological significance of each activity? Assignments: 2. De Lucia & Cairns (1969) Nature 224: 1164-1166 3. Konrad & Lehman (1974) Proc Natl Acad Sci USA 71: 2048-2051

  4. DNA polymerase 1. If a DNA polymerase were responsible for DNA duplication in E. coli, what would be expected when it becomes non-functional by mutation? 2. What is the purpose of isolating mutant with defective DNA polymerase? 3. What strategies or approaches could be used to select mutant with defective DNA polymerase?

  5. RNA polymerase 1. What is the physiological role of RNA polymerase in vivo? -- Is it essential in growth? -- How could one isolate mutant for an essential gene? 2. What is the composition of RNA polymerase? 3. What is the relationship between rifampicin-resistance and RNA polymerase? Assignment: 4. Austin et al. (1971) Nature New Biol 232: 133-136

  6. rifampicin rifapentin rifabutin rifamexyl Rifamycin: from Streptomyces mediterranei sp. Remains major antibiotics against tuberculosis; except rif-r strains arising at high frequency Artsimovitch et al (2005) Cell 122: 351

  7. Mechanism of Rif cytotoxicity strongly inhibit short RNA synthesis, but not affect transcription elongation complexes, by binding RNA polymerase b subunit with high affinity Mutations in rpoB gene giving rise to rif resistance Campbell et al (2001) Cell 104: 91

  8. Which gene is affected in rif-r strain of E. coli? What is different between rif-r and rif-null strain? What properties does a merodiploid rif-s/rif-r have? How to prevent F’ from being lost? How to prevent F’ from becoming Hfr? How to make F’ disappear? • What properties do these F’ have? • F14 • F14Su7 • KLF10

  9. Transcriptional regulation 1. What constitutes the lac operon? 2. What differs between structural genes and regulatory genes? 3. What properties are expected for mutations in structural genes? In regulatory genes? 4. What differs between mutations in repressor and mutations in operator? Assignment: 5. Pardee et al. (1959) J Mol Biol 1: 165-178

  10. Suppressors & Protein Targeting

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