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 09 28 07 1 MCB 140 09 28 07 2 a cells produce a pheromone and receptor Shmoo Al Capp 1948 Li l Abner cells produce pheromone and a receptor diploid a cells produce none of the above MCB 140 09 28 07 3 MCB 140 09 28 07 4 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 Marsh and Rose diagram MCB 140 09 28 07 5 MCB 140 09 28 07 6 1 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 A 9 MCB 140 09 28 07 MCB 140 09 28 07 7 8 A 11 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 A 12 MCB 140 09 28 07 MCB 140 09 28 07 10 9 2 1 cell HML silent MCB 140 09 28 07 11 cen 2 1 a2 a1 MAT HMRa active silent MCB 140 09 28 07 12 2 Loss of silencing at the silent mating type cassettes creates a nonmater a haploid that is a and that thinks it s a diploid 2 1 cell HML A sample screen 2 1 a2 a1 MAT HMRa 1 Take haploid cells 2 Mutate them 3 Screen for those that don t mate active active Problem mating is so much more than proper silencing of mating type loci cen active Screen for silencing mutants MCB 140 09 28 07 13 The mating pheromone response MCB 140 09 28 07 14 How to screen for silencing mutants 2 1 Also see Fig A 13 a cell Thorner diagram HML MCB 140 09 28 07 15 a2 a1 MAT HMRa active silent cen silent Jeremy Thorner a2 a1 Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 09 28 07 16 How to screen for silencing mutants 2 1 HML silent cen a2 a1 2 1 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 Rine schematic MCB 140 09 28 07 17 mate to a cells Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 09 28 07 18 3 The data Question What molecular mechanisms are responsible for silencing at the mating type loci Colonies screened 675 000 Colonies that mated to a 295 Major complementation groups 4 heterochromatin formation in metazoa prostate cancer breast cancer ageing normal gene regulation in mammals silent information regulators SIR1 SIR2 SIR3 SIR4 Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 09 28 07 19 MCB 140 09 28 07 20 How can one explain the evolution of two distinct mating types in budding yeast Homework Surely a pathway could have just evolved for the fusion of two identical haploid cells MCB 140 09 28 07 21 Two mating types have evolved under selective pressure to avoid inbreeding MCB 140 09 28 07 22 Granddaughters of any given mother can switch mating type 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 09 28 07 23 MCB 140 09 28 07 24 4 Urnov AT berkeley MCB 140 09 28 07 25 2 1 cell a cell 2 1 a2 a1 MAT HMRa silent active silent 2 1 a2 a1 a2 a1 MAT HMRa HML HML cen cen MCB 140 09 28 07 26 Compaction into chromatin brings the eukaryotic genome to life 15 000x compaction 10 5 metres 1 metre MCB 140 09 28 07 27 MCB 140 09 28 07 28 The Nucleosome Core Particle 8 histones 146 bp of DNA Beads on a string MCB 140 09 28 07 29 MCB 140 09 28 07 30 5 Histones Conserved and Charged H s Lycopersicon esculentum MCB 140 09 28 07 31 MCB 140 09 28 07 32 Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast M Grunstein Fig 6 and 7 of Kayne Fig 3 kayne Kayne et al 1988 Cell 55 27 39 MCB 140 09 28 07 33 Kayne et al 1988 Cell 55 27 39 MCB 140 09 28 07 34 Acetylation of lysine in histone tail neutralizes its charge 1964 Kayne et al 1988 Cell 55 27 39 MCB 140 09 28 07 35 MCB 140 09 28 07 36 6 Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae Table 2 Reverse genetics introduce point mutations in H4 tail Johnson et al 1990 PNAS 87 6286 6290 MCB 140 09 28 07 37 Johnson et al 1990 PNAS 87 6286 6290 MCB 140 09 28 07 38 And 5 years later Sir3p and Sir4p bind H3 and H4 tails MCB 140 09 28 07 39 Houston we have a Hecht et al 1995 Cell 80 583 MCB 140 09 28 07 40 The silencers Every nucleosome in the cell has an H3 and H4 tail two of each actually Hawthorne deletion 1963 and onwards two silencers flank the mating type loci Why do the SIRs bind only where they bind MCB 140 09 28 07 41 MCB 140 09 28 07 42 7 The key question Roy Frye Pitt How do the SIRs spread from the silencer and over the mating type loci genes 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 how do the SIRs actually silence txn 2 So do humans 5 3 The bacterial proteins are enzymes and use NAD to ADP ribosylate other proteins MCB 140 09 28 07 43 MCB 140 09 28 07 44 J Denu Sir2p is a NAD dependent histone deacetylase HDAC Sir2p Tanner et al PNAS 97 14178 2000 MCB 140 09 28 07 45 Rusche L Kirchmaier A 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