E coli E lephant 1965 1966 F Jacob J Monod A Pardee D Hawthorne H Douglas Y Oshima MCB 140 11 27 06 1 Analogy and homology as tools in genetic investigation Animal Mandibular Arch ventral Mandibular Arch dorsal Hyoid Arch dorsal Shark Meckel s cartilage Palatoquadrate cartilage Hyomandibular cartiliage Amphibian Articular bone Quadrate bone Stapes Mammal Malleus Incus Stapes MCB 140 11 27 06 2 MCB 140 11 27 06 3 a cells produce a pheromone and receptor cells produce pheromone and a receptor diploid a cells produce none of the above MCB 140 11 27 06 4 Shmoo Al Capp 1948 Li l Abner MCB 140 11 27 06 5 Marsh and Rose diagram MCB 140 11 27 06 6 The phenotype of a haploid yeast cell with respect to mating is determined by transcription factors An cell produces two transcription factors Mat 1p and Mat 2p that ensure expression of specific genes including the pheromone and receptor and repress expression of a specific genes In an a cell Mat 1p and Mat 2p are not expressed and a different transcription factor is expressed Mata1p The genes are off and the a genes pheromone and receptor are on MCB 140 11 27 06 7 A 9 MCB 140 11 27 06 8 Amazing but true A wild type haploid yeast cell contains THREE copies of mating type determining genes Copy 1 the 1 and 2 genes silent Copy 2 the a1 and a2 genes also silent Copy 3 An additional copy of genes in item 1 or of the genes in item 2 but active Whichever genes are contained in copy 3 determines the mating type MCB 140 11 27 06 9 A 11 A 12 MCB 140 11 27 06 10 An easily understood workable falsehood is more useful than an incomprehensible truth MCB 140 11 27 06 11 cell HML silent cen a2 a1 MAT HMRa active silent MCB 140 11 27 06 12 Loss of silencing at the silent mating type cassettes creates a nonmater a haploid that is a and that thinks it s a diploid cell HML active cen a2 a1 MAT HMRa active active MCB 140 11 27 06 13 Screen for silencing mutants A 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 MCB 140 11 27 06 14 The mating pheromone response Also see Fig A 13 Jeremy Thorner Thorner diagram MCB 140 11 27 06 15 How to screen for silencing mutants a cell HML silent a2 a1 a2 a1 MAT HMRa active silent cen Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 11 27 06 16 How to screen for silencing mutants HML silent cen a2 a1 mata1 1 HML active silent Note mata1 1 is a special allele of the a gene it is recessive to Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 11 27 06 17 Rine schematic mate to a cells Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 11 27 06 18 The data Colonies screened 675 000 Colonies that mated to a 295 Major complementation groups 4 silent information regulators SIR1 SIR2 SIR3 SIR4 Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 11 27 06 19 Question What molecular mechanisms are responsible for silencing at the mating type loci heterochromatin formation in metazoa prostate cancer breast cancer ageing normal gene regulation in mammals MCB 140 11 27 06 20 Homework MCB 140 11 27 06 21 How 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 MCB 140 11 27 06 22 Two mating types have evolved under selective pressure to avoid inbreeding One evolutionary advantage of mating is the production of novel genotypic combinations via the fusion of two genomes with different life histories D1 D1 x M D2 D2 MCB 140 11 27 06 23 Granddaughters of any given mother can switch mating type MCB 140 11 27 06 24 MCB 140 11 27 06 25 urnov berkeley edu MCB 140 11 27 06 26 cell a cell a2 a1 MAT HMRa silent active silent a2 a1 a2 a1 MAT HMRa HML HML cen cen MCB 140 11 27 06 27 Epigenetic inheritance In an strain the genetic information at MAT and at HML is 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 MCB 140 11 27 06 28 Compaction into chromatin brings the eukaryotic genome to life 15 000x compaction 1 metre 10 5 metres MCB 140 11 27 06 29 Beads on a string MCB 140 11 27 06 30 The Nucleosome Core Particle 8 histones 146 bp of DNA MCB 140 11 27 06 31 Histones Conserved and Charged H s Lycopersicon esculentum MCB 140 11 27 06 32 MCB 140 11 27 06 33 Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast M Grunstein Fig 3 kayne Kayne et al 1988 Cell 55 27 39 MCB 140 11 27 06 34 Fig 6 and 7 of Kayne Kayne et al 1988 Cell 55 27 39 MCB 140 11 27 06 35 Kayne et al 1988 Cell 55 27 39 MCB 140 11 27 06 36 Acetylation of lysine in histone tail neutralizes its charge 1964 MCB 140 11 27 06 37 Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae Reverse genetics introduce point mutations in H4 tail Johnson et al 1990 PNAS 87 6286 6290 MCB 140 11 27 06 38 Table 2 Johnson et al 1990 PNAS 87 6286 6290 MCB 140 11 27 06 39 MCB 140 11 27 06 40 And 5 years later Sir3p and Sir4p bind H3 and H4 tails Hecht et al 1995 Cell 80 583 MCB 140 11 27 06 41 Houston 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 MCB 140 11 27 06 42 The silencers Hawthorne deletion 1963 and onwards two silencers flank the mating type loci MCB 140 11 27 06 43 The key question How do the SIRs spread from the silencer and over the mating type loci genes how do the SIRs actually silence txn MCB 140 11 27 06 44 Roy Frye Pitt Characterization of five human cDNAs with homology to the yeast SIR2 gene Sir2 like proteins sirtuins metabolize NAD and may have protein ADPribosyltransferase activity BBRC 260 273 1999 1 Bacteria have proteins homologous to Sir2 2 So do humans 5 3 The bacterial proteins are enzymes …
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