1965 F Jacob J Monod A Pardee D Hawthorne H Douglas Y Oshima E coli E lephant 1 3 MCB 140 11 27 06 MCB 140 11 27 06 1966 Meckel s cartilage Articular bone Malleus Shark Amphibian Mammal Incus Stapes Stapes MCB 140 11 27 06 Hyomandibular cartiliage Palatoquadrate cartilage Quadrate bone Hyoid Arch dorsal Mandibular Arch dorsal MCB 140 11 27 06 diploid a cells produce none of the above cells produce pheromone and a receptor a cells produce a pheromone and receptor Mandibular Arch ventral Animal Analogy and homology as tools in genetic investigation 4 2 1 MCB 140 11 27 06 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 The phenotype of a haploid yeast cell with respect to mating is determined by transcription factors MCB 140 11 27 06 Shmoo Al Capp 1948 Li l Abner 7 5 A 9 Marsh and Rose diagram MCB 140 11 27 06 MCB 140 11 27 06 8 6 2 9 MCB 140 11 27 06 11 An easily understood workable falsehood is more useful than an incomprehensible truth MCB 140 11 27 06 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 Amazing but true cell silent HML 2 1 A 12 cen A 11 active MAT 2 1 MCB 140 11 27 06 12 silent HMRa a2 a1 MCB 140 11 27 06 10 3 active HML Jeremy Thorner Also see Fig A 13 The mating pheromone response cell 2 1 MAT active Thorner diagram cen 2 1 MCB 140 11 27 06 15 MCB 140 11 27 06 13 active HMRa a2 a1 Loss of silencing at the silent mating type cassettes creates a nonmater a haploid that is a and that thinks it s a diploid MCB 140 11 27 06 14 silent HML cen active MAT a2 a1 Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 a cell 2 1 MCB 140 11 27 06 16 silent HMRa a2 a1 How to screen for silencing mutants Problem mating is so much more than proper silencing of mating type loci 1 Take haploid cells 2 Mutate them 3 Screen for those that don t mate A sample screen Screen for silencing mutants 4 active silent HML 2 1 Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 SIR1 SIR2 SIR3 SIR4 silent information regulators Colonies screened 675 000 Colonies that mated to a 295 Major complementation groups 4 The data Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 MCB 140 11 27 06 19 MCB 140 11 27 06 17 Note mata1 1 is a special allele of the a gene it is recessive to silent mata1 1 HML cen a2 a1 2 1 How to screen for silencing mutants MCB 140 11 27 06 18 heterochromatin formation in metazoa prostate cancer breast cancer ageing normal gene regulation in mammals MCB 140 11 27 06 20 What molecular mechanisms are responsible for silencing at the mating type loci Question Jasper Rine and Ira Herskowitz 1987 Genetics 116 9 22 mate to a cells Rine schematic 5 MCB 140 11 27 06 21 M D1 D2 MCB 140 11 27 06 23 D2 x D1 One evolutionary advantage of mating is the production of novel genotypic combinations via the fusion of two genomes with different life histories Two mating types have evolved under selective pressure to avoid inbreeding Homework MCB 140 11 27 06 22 MCB 140 11 27 06 24 Granddaughters of any given mother can switch mating type Surely a pathway could have just evolved for the fusion of two identical haploid cells How can one explain the evolution of two distinct mating types in budding yeast 6 a cell cell HML cen MAT a2 a1 2 1 MAT active cen 2 1 silent HML 2 1 MCB 140 11 27 06 27 HMRa a2 a1 silent HMRa a2 a1 MCB 140 11 27 06 25 MCB 140 11 27 06 26 MCB 140 11 27 06 28 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 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 inheritance urnov berkeley edu 7 MCB 140 11 27 06 29 5metres 10 MCB 140 11 27 06 31 The Nucleosome Core Particle 8 histones 146 bp of DNA 1 metre 15 000x compaction Compaction into chromatin brings the eukaryotic genome to life H s Lycopersicon esculentum Histones Conserved and Charged Beads on a string MCB 140 11 27 06 32 MCB 140 11 27 06 30 8 Kayne et al 1988 Cell 55 27 39 Fig 6 and 7 of Kayne MCB 140 11 27 06 35 MCB 140 11 27 06 33 Kayne et al 1988 Cell 55 27 39 Kayne et al 1988 Cell 55 27 39 Fig 3 kayne MCB 140 11 27 06 36 MCB 140 11 27 06 34 Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast M Grunstein 9 Johnson et al 1990 PNAS 87 6286 6290 Table 2 MCB 140 11 27 06 39 MCB 140 11 27 06 37 Acetylation of lysine in histone tail neutralizes its charge 1964 Johnson et al 1990 PNAS 87 6286 6290 MCB 140 11 27 06 40 MCB 140 11 27 06 38 Reverse genetics introduce point mutations in H4 tail Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae 10 MCB 140 11 27 06 41 MCB 140 11 27 06 43 Hawthorne deletion 1963 and onwards two silencers flank the mating type loci The silencers Hecht et al 1995 Cell 80 583 Sir3p and Sir4p bind H3 and H4 tails And 5 years later how do the SIRs actually silence txn How do the SIRs spread from the silencer and over the mating type loci genes The key question MCB 140 11 27 06 44 MCB 140 11 27 06 42 Why do the SIRs bind only where they bind Every nucleosome in the cell has an H3 and H4 tail two of each actually Houston we have a 11 Rusche L Kirchmaier A Rine J 2002 Mol Biol Cell 13 2207 MCB 140 11 27 06 47 MCB …
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