Handout 18 - Genetic Analysis in Yeast - MC 252 Part 2 B C...

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MCB 252, Part 2 Cell cycle II Genetic analysis in yeast Budding yeast life cycle cdc mutants, CDC28 Fission yeast cloning of cdc2, regulators How does this all fit together?
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Genetic Analysis of Bacteriophage T4 Assembly Pollard and Earnshaw, 5-15 and 5-16 Biochemical and Genetic Pathway
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Concepts and Terminology Genotype/Phenotype Wild-type Allele – different forms of genes Loss-of-Function/Gain-of-Function Mutation Recessive(mostly loss-of-function) Dominant mutations (mostly gain-of-function) Pathways Genetic and Biochemical Haploid/Diploid
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Life Cycle of the Budding Yeast S. cerevisiae
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Genetic Analysis of the Cell Cycle in Budding Yeast ( S. cerevisiae) Lee Hartwell 2001 Nobel Prize Advantages for studying yeast: Rapid growth- 90 min. cell cycle Powerful genetic tools Both haploids and diploids undergo mitosis Isolaterecessivemutants in haploid
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Budding Yeast Cell Morphology Correlates with Position in Cell Cycle
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“Plating” Single Yeast Cells “Plate” Single cells Grow to colony “Rich media” WT cells
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Master Plate
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Hunting for Mutants “Rich media” Colonies Mutagenize Single cells WT cells WT cells “Uninteresting” mutants “Interesting” mutants Do Your Screen in Haploids Typical screen ~ 100,000 colonies If you are interested in the cell cycle, what phenotypedo you screen for?
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Cells defective in genes required for cell cycle progression should not be able to divide; these types of mutations will be lethal. How can one screen for (and maintain) lethal mutants in a haploid organism? Answer: screen for conditional mutations. Problem:
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Conditional Mutations Temperature SensitiveMutation WT Temp. sens. Mutation L.O.F. mutation Folded/active Folded/active Folded/active Unfolded/inactive Missing or inactive Missing or inactive 23C 37C
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Screen for mutants with defects in cell cycle Preimary screen: look for essential genes Cells defectivein cell cycleshould not be able to divide; this will be lethal. So, how do you screen for lethal mutants?
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