QuestionIf you think about it …Slide 3Slide 4The resultA distinction between genetics and biochemistrySlide 7Let’s mate“Suppression of and complementation among mutants of the regulatory gene of the lactose operon of Escherichia coli.” Bourgeois S, Cohn M, Orgel LE. JMB 14: 300 (1965)In their own wordsSlide 11Slide 12Slide 13Yeast ferment all available sugar even in the presence of oxygenA major evolutionary conservation of cellular response to sugar in the mediumAnalogy and homology as tools in genetic investigationSlide 17First experiment (Howard Douglas, 1963)What grewConclusion (ta-daaa!)Nothin’ but netButScreen for gal cellsSlide 24Slide 25Initial analysis of mutantsWhat came out“When genes are linked, PDs exceed NPDs”Linkage analysisConclusionsWhat does whatThe fun part of this complete breakfastTa-daaa!!Summary ctd.But still1966: got it!- galactose+ galactose- galactose in gal81 strainwrongGal4p is synthesized at all times, irrespective of the presence of galactoseSlide 42Slide 43Slide 44Epistasis (1982-84)Slide 46Slide 47How genes respond to environmental stimuliMore from Dr. JacobNext timeFurther reading1MCB 140 – 11/8/2006QuestionWhat is the chemical nature of the repressor?2MCB 140 – 11/8/2006If you think about it …•The repressor has to directly bind to a specific DNA sequence (the operator) in the E. coli genome.•From a reverse-engineering perspective, the simplest way to design something that interacts with DNA sequence-specifically is to use a nucleic acid that is complementary to that DNA!3MCB 140 – 11/8/2006•In 1959, a biochemical experiment was done “proving” that the lac repressor is not a protein, and is most likely an RNA molecule•In 1965, a genetic experiment was done proving that biochemical experiment entirely wrong4MCB 140 – 11/8/20061959:(When the z+ gene and the i+ gene arrive in an i- cytoplasm (no repressor), the z+ gene becomes active, and stays active for about 2 hours. At that point, the repressor is made, and shuts the z+ gene off.This offers an elegant opportunity to determine the biochemical nature of the repressor: add an inhibitor of protein synthesis, and let the cells spend their first two hours post-mating in that good stuff.A.B. Pardee and L.S. Prestidge BBA 36: 545.If the repressor is a protein, then inhibiting its synthesis following transfer of z+ into an i- cytoplasm should allow for constitutive synthesis of -galactosidase!5MCB 140 – 11/8/2006The result•Inhibiting protein synthesis does not destroy the “plateau” effect: -gal still goes off!•Conclusion: “… the repressor probably is not a protein, since it was made when [protein] synthesis was inhibited. Ribonucleic acid would seem a likely candidate for the role of the repressor.”A.B. Pardee and L.S. Prestidge BBA 36: 545.6MCB 140 – 11/8/2006A distinction between genetics and biochemistry•The validity of Pardee’s conclusion is unequivocally dependent on a biochemical phenomenon (total inhibition of protein synthesis by 5-methyltryptophan)•It turns out that not all protein synthesis in E. coli is inhibited by 5-me-T…•A genetic experiment (=crosses between strains of different genotype) was performed to prove Pardee wrong7MCB 140 – 11/8/20068.288MCB 140 – 11/8/2006Let’s mate1. Make an i- strain of E. coli (constitutive)2. Make several such strains (=different kinds of mutations)3. Mate those i- E. coli to other E. coli carrying nonsense suppressor tRNA genes4. See what happens.9MCB 140 – 11/8/2006“Suppression of and complementation among mutants of the regulatory gene of the lactose operon of Escherichia coli.”Bourgeois S, Cohn M, Orgel LE.JMB 14: 300 (1965)1. Cross an i- strain with a tRNAsup strain2. Measure activity of -gal (+ or - lactose in the medium).Suppressor: none (wt) su1 su3lactose no yes no yes no yesStraini+ 8 4400 6 4400 14 3900i- 7600 7000 1200 1550 3400 710010MCB 140 – 11/8/2006In their own words“We know … that the suppressor strains used here act at the level of translation by allowing the chain-terminating codon to be read as an amino acid. We have shown suppression of i- mutations by these suppressors and conclude therefore that the i gene of the lactose operon codes for a protein.”11MCB 140 – 11/8/2006Regulation of genes occurs via the interaction of trans-acting factors (proteins) with cis-acting sequences near the genes themselves.+stimulus+12MCB 140 – 11/8/2006François Jacob:“If it’s true for E. coli, it must be true for E. lephant.”13MCB 140 – 11/8/2006Budding (brewer’s and baker’s) yeast, Saccharomyces cerevisiae14MCB 140 – 11/8/2006Yeast ferment all available sugar even in the presence of oxygenMammals: sugar pyruvate CO2+H2OS.c.: sugar pyruvate C2H5OH+CO2glycolysisglycolysisrespirationTCA+O.P.fermentation15MCB 140 – 11/8/2006A major evolutionary conservation of cellular response to sugar in the mediumIn Saccharomyces cerevisiae (budding yeast = a fungus = a eukaryote): 1. Enzymes for utilization of the sugar galactose are induced ~1000-fold by galactose.2. These enzymes are also severely repressed by glucose in the medium.3. Thus, for these genes to be induced fully, the medium must contain galactose and no glucose.Just like E. coli.16MCB 140 – 11/8/2006Analogy and homology as tools in genetic investigationAnimalMandibular Arch (ventral)Mandibular Arch (dorsal)Hyoid Arch(dorsal)Shark Meckel's cartilagePalatoquadrate cartilageHyomandibular cartiliageAmphibian Articular (bone) Quadrate (bone) StapesMammal Malleus Incus Stapes17MCB 140 – 11/8/200618MCB 140 – 11/8/2006First experiment(Howard Douglas, 1963)Goal: make i- yeast – that is, yeast that synthesize galactose-metabolizing enzymes constitutively.1. Take mutant strain (gal3) that has a markedly delayed response to galactose and does not grow on it very well at all.2. Grow on galactose – see what grows.Douglas HC, Penroy G (1963) A gene controlling inducibility of the galactose pathway enzymes in Saccharomyces. Biochim Biophys Acta 68: 155.19MCB 140 – 11/8/2006What grew1. One would expect revertants of the gal3 mutation. Those didn’t show up.2. What did show up was true i- cells – yeast that synthesized the GAL enzymes constitutively (that’s why they grew).3. They made i+ / i- cells – they were inducible.20MCB 140 – 11/8/2006Conclusion (ta-daaa!)“The inducibility in gene in yeast fits the description proposed by Monod and Jacob for regulator genes. … By analogy
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