11MCB 140 11/27/06E. coli = E. lephant ?F. JacobJ. MonodA. PardeeD. HawthorneH. DouglasY. Oshima1965 19662MCB 140 11/27/06Analogy and homology as tools in genetic investigationStapesIncusMalleusMammalStapesQuadrate (bone)Articular (bone)AmphibianHyomandibular cartiliagePalatoquadrate cartilageMeckel's cartilageSharkHyoid Arch(dorsal)Mandibular Arch (dorsal)Mandibular Arch (ventral)Animal3MCB 140 11/27/064MCB 140 11/27/06a cells produce a pheromone and α receptorα cells produce α pheromone and a receptordiploid (a/α) cells produce none of the above25MCB 140 11/27/06ShmooAl Capp (1948) – Li’l Abner6MCB 140 11/27/06Marsh and Rose diagram7MCB 140 11/27/06The phenotype of a haploid yeast cell with respect to mating is determined by transcription factorsAn α cell produces two transcription factors, Matα1p and Matα2p, that ensure expression of α specific genes, including the pheromone and receptor, and repress expression of a specific genes.In an a cell, Matα1p and Matα2p are not expressed, and a different transcription factor is expressed, Mata1p. The α genes are off, and the a genes (pheromone and receptor) are on.8MCB 140 11/27/06A.939MCB 140 11/27/06Amazing but trueA wild-type haploid yeast cell contains THREE copies of mating type-determining genes:• Copy #1: the α1 and α2 genes (silent).• Copy #2: the a1 and a2 genes (also silent).• Copy #3: An additional copy of genes in item 1, orof the genes in item 2, but active.Whichever genes are contained in copy #3 determines the mating type.10MCB 140 11/27/06A.11A.1211MCB 140 11/27/06“An easily understood, workable falsehood is more useful than an incomprehensible truth.”12MCB 140 11/27/06cenMATHMLαHMRaα1α2a1a2α cellα1α2active silentsilent413MCB 140 11/27/06Loss of silencing at the silent mating type cassettes creates a “nonmater” – a haploid that is a/α and that thinks it’s a diploid.cenMATHMLαHMRaα1α2a1a2α cellα1α2active activeactive14MCB 140 11/27/06Screen for silencing mutantsA sample “screen”:1. Take haploid cells.2. Mutate them.3. Screen for those that don’t mate.Problem: mating is so much more than proper silencing of mating type loci!!15MCB 140 11/27/06The mating pheromone responseJeremy ThornerThorner diagramAlso see Fig. A.13.16MCB 140 11/27/06How to screen for silencing mutantscenMATHMLαHMRaα1α2a1a2a cella1a2active silentsilentJasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.517MCB 140 11/27/06How to screen for silencing mutantscenmata1-1HMLαα1α2a1a2active silentsilentJasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.HMLαα1α2Note: mata1-1 is a special allele of the a gene – it is recessive to α18MCB 140 11/27/06Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.Rine schematicmate to a cells19MCB 140 11/27/06The data• Colonies screened: 675,000• Colonies that mated to a: 295• Major complementation groups: 4silent information regulators:SIR1, SIR2, SIR3, SIR4Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.20MCB 140 11/27/06QuestionWhat molecular mechanisms are responsible for silencing at the mating type loci?→ heterochromatin formation in metazoa→prostate cancer→ breast cancer→ ageing→ “normal” gene regulation in mammals621MCB 140 11/27/06Homework22MCB 140 11/27/06How can one explain the evolution of two distinct mating types in budding yeast?Surely a pathway could have just evolved for the fusion of two identical haploid cells?23MCB 140 11/27/06Two mating types have evolved under selective pressure to avoid inbreedingMD1D2D1D2One evolutionary advantage of mating is the production of novel genotypic combinations via the fusion of two genomes with different life histories.x24MCB 140 11/27/06Granddaughters of any given mother can switch mating type725MCB 140 11/27/0626MCB 140 11/27/[email protected] 140 11/27/06cenMATHMLαHMRaα1α2a1a2α cellα1α2cenMATHMLαHMRaα1α2a1a2a cella1a2active silentsilent28MCB 140 11/27/06Epigenetic inheritance• In an α strain, the genetic information at MAT and at HMLαis identical.• The one at MAT is expressed, but the one at HML is not – it is epigenetically silenced.Epigenetic: mitotically stable (persists through cell division) change in gene expression state that is not associated with a change in DNA sequence.Examples: X chromosome inactivation; imprinted genes; transgene silencing in gene therapy.829MCB 140 11/27/06> 1 metre< 10-5metres15,000xcompactionCompaction into chromatin brings the eukaryotic genome to life30MCB 140 11/27/06“Beads on a string”31MCB 140 11/27/06The Nucleosome Core Particle:8 histones, 146 bp of DNA32MCB 140 11/27/06Histones: Conserved and ChargedH.s. = Lycopersicon esculentum933MCB 140 11/27/0634MCB 140 11/27/06“Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast” (M. Grunstein) Kayne et al. (1988) Cell 55: 27-39.Fig. 3 kayne35MCB 140 11/27/06Kayne et al. (1988) Cell 55: 27-39.Fig. 6 and 7 of Kayne.36MCB 140 11/27/06Kayne et al. (1988) Cell 55: 27-39.1037MCB 140 11/27/06Acetylation of lysine in histone tail neutralizes its charge (1964)38MCB 140 11/27/06“Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae”Johnson et al. (1990) PNAS 87: 6286-6290 .Reverse genetics: introduce point mutations in H4 tail!!39MCB 140 11/27/06Johnson et al. (1990) PNAS 87: 6286-6290 .Table 240MCB 140 11/27/061141MCB 140 11/27/06And 5 years later …Sir3p and Sir4p bind H3 and H4 tailsHecht et al. (1995) Cell 80: 583.42MCB 140 11/27/06Houston, we have a …Every nucleosome in the cell has an H3 and H4 tail (two of each, actually).Why do the SIRs bind only where they bind?43MCB 140 11/27/06The silencers“Hawthorne deletion” (1963) and onwards:two silencers flank the mating type loci:44MCB 140 11/27/06The key questionHow do the SIRs spread from the silencer and over the mating type loci genes?= how do the SIRs actually silence txn?1245MCB 140 11/27/06Roy Frye (Pitt)“Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity” BBRC 260: 273 (1999).1. Bacteria have proteins homologous to Sir2.2. So do humans (>5).3. The bacterial proteins are enzymes, and use NAD to ADP-ribosylate other proteins. 46MCB 140 11/27/06J. Denu: Sir2p is a NAD-dependenthistone deacetylase (HDAC)Tanner
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