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Berkeley MCELLBI 140 - Lecture Notes

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Further conservationSimple experiment (1985)ConclusionThis is what this meansDeath by sugarWhat M. Carlson foundSlide 71986: SNF1 is a protein kinaseSearch for something, part IWhat was found in part ISlide 11Search for something, part IILife or deathMass action in actionWhat part II foundSlide 16They are the same gene.Slide 18Slide 19How genes respond to environmental stimuliMore from Dr. JacobMCB 140 11-20-06 1Further conservationInduction of GAL genes by galactose is only half the story.The GAL genes are also severely repressed by glucose (the same is true of the lac genes in E. coli). In fact, all non-glucose sugar utilization circuits in yeast are repressed by glucose.MCB 140 11-20-06 2Simple experiment (1985)GAL1 promoter fragmentHIS3 promoterHIS3Struhl K. Nature 317: 822 (1985).+ glucoseTranscription of HIS3 gene is repressed, even under conditions when the HIS3 promoter is maximally active!(amino acid starvation)MCB 140 11-20-06 3ConclusionNormally, his3 expression … occurs at a basal level which can be induced by conditions of amino-acid starvation. However, in glucose medium, the catabolite regulatory sequence overrides the normal his3 promoter elements and reduces transcription both in normal and starvation conditions. The implication from these results is that in contrast to catabolite repression in Escherichia coli, which is mediated by catabolite-activating protein (CAP), catabolite repression in yeast occurs by a negative control mechanism involving a putative repressor protein. The observation that this regulatory site exerts its repressing effects even when located upstream of an intact promoter region suggests that repression in yeast is not mediated by steric interference between regulatory proteins and the transcriptional apparatus.Struhl K. Nature 317: 822 (1985).MCB 140 11-20-06 4This is what this meansThere must be some REPRESSOR that is glucose-responsive – it kills sugar-utilizing enzyme gene promoters when glucose is present. Under low glucose, this repressor goes away.How to find that repressor?MCB 140 11-20-06 5Death by sugarMarian Carlson screened for mutants that would be sucrose non-fermenters (snf) – that is, would not grow on sucrose in the absence of glucose.1. Enzymes that metabolize sucrose.2. “Suc4” – the positive regulator that responds to sucrose addition (no such thing).3. The “glucose sensor” – if you mutate it, the cell will think glucose is present!Carlson M. et al. Genetics 98: 25 (1981).MCB 140 11-20-06 6What M. Carlson found1. suc2 – mutations in invertase, the key enzyme that breaks down sugar.2. snf1 – an unlinked mutation.“The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2+ and SUC7+ strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. “Carlson M. et al. Genetics 98: 25 (1981).MCB 140 11-20-06 7 Bill WattersonMCB 140 11-20-06 81986: SNF1 is a protein kinaseGlucose present = Snf1p inactiveGlucose absent = Snf1p active, phosphorylates something, and that something stops repressing the sugar-utilizing gene promoters.The search for something DOWNSTREAM of SNF1.Celenza JL, Carlson M. Science 233: 1175 (1986).MCB 140 11-20-06 9Search for something, part IClassical epistasis screen – search for mutations that would be epistatic to SNF1.1. Take snf1 cells (do not grow on sucrose).2. Mutagenize the poor things.3. Cells that grow on sucrose contain an extragenic suppressor of the snf1 mutation.Schüller HJ, Entian KD. J. Bacteriol. 173: 2045 (1991).MCB 140 11-20-06 10What was found in part I•New gene, CAT4. •Mutations in CAT4 nicely suppressed the growth defect of snf1 cells.In other words, snf1 cat4 cells do not exhibit a sucrose nonfermentation phenotype.= Cat4p acts downstream of Snf1p.MCB 140 11-20-06 11MCB 140 11-20-06 12Search for something, part IIFind a protein the overexpression of which will shut down the GAL1 promoter. Even in the presence of galactose.High-copy suppressor screen:1. Take mutant cell.2. Transform a library of cDNAs driven by a strong promoter.3. Find cDNA that suppresses mutant phenotype.Nehlin JO, Ronne H. EMBO J. 9: 2891 (1990).MCB 140 11-20-06 13Life or deathGAL1 promoterdeath+ galactosecell rapidly diesMCB 140 11-20-06 14Mass action in actionOverexpress something that will compete with Gal4p activating the promoter:MCB 140 11-20-06 15What part II foundMIG1 (multicopy inhibitor of GAL genes).MCB 140 11-20-06 16CAT4 – epistatic to SNF1.MIG1 – silences GAL1 promoter.MCB 140 11-20-06 17They are the same gene.CAT4 = MIG1Mig1p – DNA-binding protein(transcriptional repressor)MCB 140 11-20-06 18MCB 140 11-20-06 19MCB 140 11-20-06 20How genes respond to environmental stimuliMCB 140 11-20-06 21More from Dr. Jacob“I have always been convinced that the same principles operating in bacteria are also operating in higher organisms with added complexity. The question therefore is to understand what kind of complexity is involved and how it is


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Berkeley MCELLBI 140 - Lecture Notes

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