1MCB 140 11/3/05 1“Influential ideas are always simple. Since natural phenomena need not be simple, we master them, if at all, by formulating simple ideas and exploring their limitations.”Al HersheyMCB 140 11/3/05 2MCB 140 11/3/05 3 MCB 140 11/3/05 4MCB 140 11/3/05 5“Bacterial” genetics IMCB 140 11/3/05 6“What are the genes? What is the nature of the elements of heredity that Mendel postulated as purely theoretical units? … Frankly, these are questions with which the working geneticist has not much concern himself… If the gene is a material unit, it is a piece of a chromosome; if it is a fictitious unit, it must be referred to a definite location in a chromosome. … Therefore, it makes no difference in the actual work in genetics which point of view is taken.”T.H. Morgan The Relation of Genetics to Physiology and MedicineNobel Lecture, June 4, 193427MCB 140, 2/25/05DNA ! RNA ! proteincentral dogma of molecular biologyMCB 140 11/3/05 87.17MCB 140 11/3/05 9Max Delbrück (1906 -1981) & Salvador Luria (1912 - 1991), Cold Spring Harbor Laboratory, Long Island NY, Summer 1941 MCB 140 11/3/05 10Today even the layman thinks of resistant bacteria as originating from mutation … but when Luria and Delbrück first got together, conventional bacteriologists were by no means clear that microorganisms could be tought about genetically… Many believed that resistance was some kind of adaptation induced, in a few of the bacteria in a culture, by the exposure to the antibacterial agent.Judson p. 55MCB 140 11/3/05 11The idea smacks of the pre-Mendelian, pre-Darwinian notion of the inheritance of acquired characteristics; Luria damned bacteriology as the “last stronghold of Lamarckism.”Judson p. 55MCB 140 11/3/05 12Let’s all do science in NevadaOne Saturday evening … Luria went to a faculty dance… There, watching the fluctuating returns obtained by colleagues gambling on a slot machine, he thought of the experiment that would distinguish between resistance induced in bacteria and resistance resulting from previous spontaneous mutation upon which selection acts. Judson p. 553MCB 140 11/3/05 13What Luria perceived was that previous spontaneous mutation would pay out jackpots of resistant bacteria that would fluctuate much more widely in size than those paid out by induction. He tried the first experiment on the following morning and wrote off to Delbrueck; Delbrueck promptly replied that Luria really ought to go to church …Judson p. 55MCB 140 11/3/05 147.4MCB 140 11/3/05 15What Luria actually didSample set A:1. Inoculate bacteria into individual cultures (1 bacterium per culture).2. Let it grow up to a large number.Sample set B:1. Take an aliquot of bacteria, and start a culture (which will therefore not be clonal).2. Let them grow up to a large numberExpose both to phage, and count, how many phage-resistant colonies per culture are found. Ask, if there is a difference between these two sample sets.MCB 140 11/3/05 16S. Luria, M. Delbrück (1943)Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28: 491-511.“If the production of resistance began only at the moment of exposure to phage, then it wouldn’t matter whether the bacteria came from many individual cultures or one bulk culture. … When Luria performed the experiment, though, the twenty separate cultures showed much wider fluctuations from the average number of resistant colonies, indicating that a few of the individual tubes contained resistant bacteria from near the beginning of the overnight growth period.”Judson p. 56MCB 140 11/3/05 17Brock p. 59MCB 140 11/3/05 184MCB 140 11/3/05 19 MCB 140 11/3/05 20George Beadle (left) and Edward Tatum (right) receiving their Nobel PrizesRemember Prof. Amacher’s lecture #8?MCB 140 11/3/05 21 MCB 140 11/3/05 227.20MCB 140 11/3/05 23Joshua LederbergMCB 140 11/3/05 24“Conjugation” in bacteriaTake strain of E. coli that is auxotrophic for two distinct nutrients (thiamine and leucine).Take different strain of E. coli that is also auxotrophic for two distinct nutrients, but different ones (biotine and cysteine).Mix the two.Ask, if ANY NOVEL PHENOTYPES APPEAR.5MCB 140 11/3/05 2514.11J. Lederberg, E. Tatum (1946)Novel genotypes in mixed cultures of biochemical mutants of bacteria. Cold Spring Harbor Symp. Quant. Biol. 11: 113-114.MCB 140 11/3/05 26ParameciumMCB 140 11/3/05 27B.H. and A.H.In the pre-Hayes period, mating in bacteria was envisioned as a conventional sex process, perhaps modified by aspects of “relative sexuality,” but nevertheless a standard haploid/diploid/meiosis mechanism. After Hayes, it was known that bacteria were not just small cells, but constituted a completely different kind of cell … The terms prokaryote and eukaryote were not introduced until 1962. Brock p. 87MCB 140 11/3/05 28William HayesMCB 140 11/3/05 29Selman WaksmanstreptomycinMCB 140 11/3/05 30Hayes expt:Take strain A, which is streptomycin-resistant, and auxotrophic for biotin and methionine.Take strain B, which is streptomycin-sensitive, and auxotrophic for threonine and leucine.Mix the two on a minimal-medium plate containing streptomycin.Wait and see.6MCB 140 11/3/05 31Rich husband, poor wife is not the same as poor husband, rich wifeCross #1:Strain A (StrR, B-, M-) × Strain B (StrS, L-, T-)Result: streptomycin completely inhibits prototroph formation (i.e., appearance of B+,M+,L+,T+ bacteria) if added before conjugation is complete.Cross #2:Strain A (StrS, B-, M-) × Strain B (StrR, L-, T-)Result: streptomycin has no effect whatsoever. You can add it all you want, at any time, and prototrophs will still form!!MCB 140 11/3/05 32“'I discussed these results with Denny Mitchison and I think it was he who first suggested that one of the parents, A, might be acting as a gene donor and the other, B, as a recipient'. It was from this experiment that the concept of asymmetry in bacterial sexuality arose. Parent B was the recipient or 'female', the continued viability of which was essential for the whole process of recombination and segregation, while the A donor or 'male' cell was dispensable once genetic transfer had been effected.”W. Hayes (1952) Recombination in Bact. coli K 12; unidirectional transfer of genetic material. Nature 169: 118.Brock p. 89MCB 140 11/3/05 33 MCB 140 11/3/05 3414.12MCB 140 11/3/05 3514.12MCB 140 11/3/05 3614.137MCB 140 11/3/05 3714.14MCB 140 11/3/05 38E. Wollman, F. Jacob (1955) Sur le
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