“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 “Bacterial” genetics 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 and Nobel Lecture, June 4, 1934

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DNA Æ RNA Æ protein

central dogma of molecular biology

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Today 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.

Max Delbrück (1906 -1981) & (1912 - 1991), Cold Spring Harbor Laboratory, Long Island NY, Summer 1941 Judson p. 55 MCB 140 09-19-08 5 MCB 140 09-19-08 6

1 The idea smacks of the pre- Mendelian, pre-Darwinian Let’s all do science in Nevada notion of the inheritance of acquired characteristics; One Saturday evening … Luria went to Luria damned bacteriology a faculty dance… There, watching the fluctuating returns obtained by as the “last stronghold of colleagues gambling on a slot Lamarckism.” 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.

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What 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 …

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S. Luria, M. Delbrück (1943) What Luria actually did Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28: 491-511. Sample set A: Sample set B: “If the production of resistance began only at the 1. Inoculate bacteria 1. Take an aliquot of moment of exposure to phage, then it wouldn’t into individual bacteria, and start a matter whether the bacteria came from many cultures (1 culture (which will individual cultures or one bulk culture. … When bacterium per therefore not be Luria performed the experiment, though, the culture). clonal). twenty separate cultures showed much wider 2. Let it grow up to a 2. Let them grow up to fluctuations from the average number of resistant large number. a large number colonies, indicating that a few of the individual tubes contained resistant bacteria from near the Expose both to phage, and count, how many phage- beginning of the overnight growth period.” resistant colonies per culture are found. Ask, if there is a Judson p. 56 difference between these two sample sets. MCB 140 09-19-08 11 MCB 140 09-19-08 12

2 Brock p. 59 MCB 140 09-19-08 13 MCB 140 09-19-08 14

George Beadle (left) and (right) receiving their Nobel Prizes

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Beadle and Tatum: the rationale In 1940, while teaching at Stanford, Tatum reviewed the nutritional-growth-factor requirements that distinguish related bacterial species. This raised the question of whether mutations can lead to nutritional requirements in species that do not already require a growth factor. As Beadle was already familiar with Neurospora from his contact with Dodge and Lindegren, he recognized it as an ideal with which to pursue this question. First, Neurospora could be grown on a simple minimal medium that contained inorganic salts, sucrose and a single vitamin. The idea of imposing further nutritional requirements by mutation was plausible. Moreover, Neurospora cultures were haploid, and therefore recognition of recessive, loss-of- function mutations should be straightforward. And most importantly, Neurospora had orthodox Mendelian genetics, an attribute that would be vital in a continuing dispute about the role of genes — the idea still persisted among embryologists that the fundamental information regarding body plan, organ systems and the 'epigenetic' features of development lay in the cytoplasm. Beadle and Tatum did their experiments in part to convince many sceptical biologists that genes control the fundamental processes of life, and not just the final touches of development, such as wing shape or eye pigment. To show this, it was important to use a eukaryote that was simpler than Drosophila and to focus on metabolic functions that could not possibly be considered as final touches.

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3 Beadle and Tatum: Thirty points on the midterm, the beginnings so wake up "Observing him [Tatum] writing sequences of reactions on the blackboard, I suddenly realized how stupid we had been all these years. Here were all those enzymatic reactions already worked out by competent . If our gene– concepts were correct, then we ought to be able to identify the genes immediately responsible for specifically known enzyme-catalysed reactions. So why not reverse the approach? Instead of looking for reactions by controlled by known genes, why not look for genes that control already known chemical reactions? We might then expect to find mutations ... characterized by an inability to synthesize essential diffusible substances such as vitamins, amino acids and other building blocks of the cell's protoplasm."

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Ka-BOOOM

It took Beadle and Tatum only 5 months to find the first 3 nutritional mutants in their irradiated cultures; one required pyridoxin, another needed p-aminobenzoic acid and the third required thiamin. The first public announcement of their accomplishment was at a Caltech seminar where Beadle went to recruit people for his research group. Not surprisingly, his news was a bombshell. Norman H. Horowitz, one of the people he recruited, recalls it clearly: The talk lasted only half an hour, and when it was suddenly over, the room was silent. The silence was a form of tribute. The audience was thinking nobody with such a discovery could stop speaking after just 30 minutes — there must be more. Superimposed on this thought was the realization that something historic had happened. Each one of us, I suspect, was surveying, as best he could, the consequences of the revolution that had just taken place."

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Joshua Lederberg “Conjugation” in bacteria

Take 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.

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4 J. Lederberg, E. Tatum (1946) Novel genotypes in mixed cultures of biochemical mutants of bacteria. Cold Spring Harbor Symp. Quant. Biol. 11: 113-114.

Paramecium

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B.H. and A.H. William Hayes

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.

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Selman Waksman Hayes expt: Take strain A, which is -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.

streptomycin

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5 Rich husband, poor wife “'I discussed these results with Denny Mitchison and I think it was he who first suggested that one is not the same as poor husband, rich wife of the parents, A, might be acting as a gene Cross #1: donor and the other, B, as a recipient'. Strain A (StrR, B-, M-) × Strain B (StrS, L-, T-) It was from this experiment that the concept of Result: streptomycin completely inhibits prototroph asymmetry in bacterial sexuality arose. Parent B formation (i.e., appearance of B+,M+,L+,T+ was the recipient or 'female', the continued bacteria) if added before conjugation is viability of which was essential for the whole complete. process of recombination and segregation, while Cross #2: the A donor or 'male' cell was dispensable once Strain A (StrS, B-, M-) × Strain B (StrR, L-, T-) genetic transfer had been effected.” Result: streptomycin has no effect whatsoever. W. Hayes (1952) Recombination in Bact. coli K 12; You can add it after the onset of conjugation, yet unidirectional transfer of genetic material. Nature prototrophs will still form!! 169: 118.

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Fig. 15.12

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6 Fig. 15.15

E. Wollman, F. Jacob (1955) Sur le mecanisme du transfert de materiel genetique au cours de la recombinaison chez E. coli K12. CR Academie Sciences 240: 2449.

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Cours de Physiologie et de génétique bactériennes, 1957 Illustration humoristique des cours par une série de dessins de F. Lavallé Légendes de Georges Cohen The life cycle of a temperate phage http://www.pasteur.fr/infosci/archives/mon/im_laval.html

Interruption de la pénétration de l'ADN mâle dans le cytoplasme de la bactérie femelle à l'aide d'un warring blender (simple mixer ménager). Fig. 15.21 MCB 140 09-19-08 41 MCB 140 09-19-08 42

7 Three aspects of phage biology with long-term impact 1. Transduction (phage carrying additional genetic information from cell to cell) → oncoretroviruses 2. Lysogeny (phage resident in bacterial genome) → latent viruses in eukaryotic genomes 3. Recombination between phage → the fine structure of a gene

Annual budget: $3,780.00

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8 Bronfenbrenner

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Further reading

Horace Judson The Eighth Day of Creation

Thomas Brock The Emergence of Bacterial Genetics

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