topic 15_rivier_252 - MCB 252 Topic 15 Cell Cycle: Genetic...

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Unformatted text preview: MCB 252 Topic 15 Cell Cycle: Genetic Analysis of the Yeast Cell Cycle Reading: Lodish 20.3 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? Genetic Analysis of Phage T4 Assembly Pollard and Earnshaw, 5-15 and 5-16 A Biochemical and Genetic Pathway Concepts and Terminology Genotype/Phenotype Wild-type Allele Loss-of-Funtion Mutation/Gain-of-Function Mutation Recessive/Dominant Pathways -> Genetic and Biochemical Life Cycle of the Budding Yeast S. cerevisiae 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 Isolate recessive mutants in haploid Budding Yeast Cell Morphology Correlates with Position in Cell Cycle Plating Single Yeast Cells Plate Single cells Grow to colony Rich media WT cells Master Plate 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 phenotype do you screen for? 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: Conditional Mutations Temperature Sensitive Mutation WT ts Mutation LOF mutation Folded/active Folded/active Folded/active Unfolded/inactive Missing or inactive Missing or inactive 23C 37C Screen for mutants with defects in cell cycle Preimary screen: look for essential genes Cells defective in cell cycle should not be able to divide; this will be lethal. So, how do you screen for lethal mutants? 4 3 plate individual aliquots incubate at 23C Lodish, et al., Fig. 5- 6 screen for conditional mutants Two Stages of the Cell Division Cycle (CDC) Screen 1. Screen for ts mutations in essential genes but lots of genes are essential, how do we identify the genes that are required for specific events in the cell cycle? 2. Secondary screen -> search among the ts mutant collection for genes that cause arrest at specific stages of the cell cycle. Terminal Phenotypes: Arrest at a Single Point in the Cell Cycle How does one identify cell cycle mutants? Secondary screen: look at terminal phenotype Permissive Temperature Restrictive Temperature Cells proceed unaffected through cell cycle Cells arrest at one point in cell cycle Cell cycle is arrested at multiple random times in the cycle time www.ergito.com great experiments 8.4 cdc = cell division cycle mutants; functions required at specific points in the cell cycle Budding Yeast CDC Mutants (> 50) cdc28 Terminal Phenotypes cdc7 cdc9 cdc15 CDC15 Arrest Point Assynchronous Population cdc15 terminal phenotype cell division cycle...
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topic 15_rivier_252 - MCB 252 Topic 15 Cell Cycle: Genetic...

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