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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 α receptor Shmoo Al Capp (1948) – Li’l Abner α cells produce α pheromone and a receptor diploid (a/α) 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 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. 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 α1 and α2 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 α strain, the genetic information at MAT and at HMLα 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 α2 α1 α cell HMLα silent MCB 140 09-28-07 11 cen α2 α1 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/α and that thinks it’s a diploid. α2 α1 α cell HMLα A sample “screen”: α2 α1 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 α2 α1 Also see Fig. A.13. a cell Thorner diagram HMLα 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 α2 α1 HMLα silent cen a2 a1 α2 α1 mata1-1 HMLα active silent Note: mata1-1 is a special allele of the a gene – it is recessive to α 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 α2 α1 α cell a cell α2 α1 a2 a1 MAT HMRa silent active silent α2 α1 a2 a1 a2 a1 MAT HMRa HMLα HMLα 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, Rine J (2002) Mol. Biol. Cell 13: 2207. MCB 140 09-28-07 46 Histone tail acetylation promotes chromatin unfolding (somehow) acetylation MCB 140 09-28-07 47 8