11MCB 140 09-28-07Analogy and homology as tools in genetic investigationStapesIncusMalleusMammalStapesQuadrate (bone)Articular (bone)AmphibianHyomandibular cartiliagePalatoquadrate cartilageMeckel's cartilageSharkHyoid Arch(dorsal)Mandibular Arch (dorsal)Mandibular Arch (ventral)Animal2MCB 140 09-28-073MCB 140 09-28-07a cells produce a pheromone and α receptorα cells produce α pheromone and a receptordiploid (a/α) cells produce none of the above4MCB 140 09-28-07ShmooAl Capp (1948) – Li’l Abner5MCB 140 09-28-07Marsh and Rose diagram6MCB 140 09-28-07The 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.27MCB 140 09-28-07A.98MCB 140 09-28-07Amazing 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.9MCB 140 09-28-07Epigenetic 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 epigeneticallysilenced.10MCB 140 09-28-07A.11A.1211MCB 140 09-28-0712MCB 140 09-28-07cenMATHMLαHMRaα1α2 a1a2α cellα1α2active silentsilent313MCB 140 09-28-07Loss of silencing at the silent mating type cassettes creates a “nonmater” – a haploid that is a/α and that thinks it’s a diploid.cenMATHMLαHMRaα1α2 a1a2α cellα1α2active activeactive14MCB 140 09-28-07Screen 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 09-28-07The mating pheromone responseJeremy ThornerThorner diagramAlso see Fig. A.13.16MCB 140 09-28-07How to screen for silencing mutantscenMATHMLαHMRaα1α2 a1a2a cella1a2active silentsilentJasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.17MCB 140 09-28-07How 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 09-28-07Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.Rine schematicmate to a cells419MCB 140 09-28-07The 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 09-28-07QuestionWhat molecular mechanisms are responsible for silencing at the mating type loci?→ heterochromatin formation in metazoa→prostate cancer→ breast cancer→ ageing→ “normal” gene regulation in mammals21MCB 140 09-28-07Homework22MCB 140 09-28-07How 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 09-28-07Two 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 09-28-07Granddaughters of any given mother can switch mating type525MCB 140 09-28-0726MCB 140 09-28-07Urnov AT berkeley27MCB 140 09-28-07cenMATHMLαHMRaα1α2 a1a2α cellα1α2cenMATHMLαHMRaα1α2 a1a2a cella1a2active silentsilent28MCB 140 09-28-07> 1 metre< 10-5metres15,000xcompactionCompaction into chromatin brings the eukaryotic genome to life29MCB 140 09-28-07“Beads on a string”30MCB 140 09-28-07The Nucleosome Core Particle:8 histones, 146 bp of DNA631MCB 140 09-28-07Histones: Conserved and ChargedH.s. = Lycopersicon esculentum32MCB 140 09-28-0733MCB 140 09-28-07“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 kayne34MCB 140 09-28-07Kayne et al. (1988) Cell 55: 27-39.Fig. 6 and 7 of Kayne.35MCB 140 09-28-07Kayne et al. (1988) Cell 55: 27-39.36MCB 140 09-28-07Acetylation of lysine in histone tail neutralizes its charge (1964)737MCB 140 09-28-07“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!!38MCB 140 09-28-07Johnson et al. (1990) PNAS 87: 6286-6290.Table 239MCB 140 09-28-0740MCB 140 09-28-07And 5 years later …Sir3p and Sir4p bind H3 and H4 tailsHecht et al. (1995) Cell 80: 583.41MCB 140 09-28-07Houston, 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?42MCB 140 09-28-07The silencers“Hawthorne deletion” (1963) and onwards:two silencers flank the mating type loci:843MCB 140 09-28-07The key questionHow do the SIRs spread from the silencer and over the mating type loci genes?= how do the SIRs actually silence txn?44MCB 140 09-28-07Roy 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. 45MCB 140 09-28-07J. Denu: Sir2p is a NAD-dependenthistone deacetylase (HDAC)Tanner et al., PNAS 97: 14178 (2000)Sir2p46MCB 140 09-28-07Rusche L, Kirchmaier A, Rine J (2002) Mol. Biol. Cell 13: 2207.47MCB 140 09-28-07acetylationHistone tail acetylation promotes chromatin unfolding
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