MCB 252 Topic 31 Cell Cycle Genetic Analysis of the Yeast Cell CycleProf David Rivier MCB 252 Spring 2015 MCB 252 Topic 15 Cell Cycle Genetic Analysis of the Yeast Cell Cycle Reading Lodish 20 3 20 5 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 Function Mutation Gain of Function Mutation Recessive Dominant Pathways Genetic and Biochemical Haploid Diploid Also recall that LOF mutations and GOF mutations often have opposite phenotypes Life Cycle of the Budding Yeast S cerevisiae Genetic Analysis of the Cell Cycle in Budding Yeast S cerevisiae Advantages for studying yeast Rapid growth 90 min cell cycle Powerful genetic tools Both haploids and diploids undergo mitosis Isolate recessive mutants in haploid Lee Hartwell 2001 Nobel Prize Budding Yeast Cell Morphology Correlates with Position in Cell Cycle Plating Single Yeast Cells Plate Single cells WT cells Grow to colony Rich media Master Plate Hunting for Mutants Mutagenize Single cells Rich media 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 Colonies Problem 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 Conditional Mutations 23C 37C WT Folded active Folded active Folded active Unfolded inactive Missing or inactive Missing or inactive ts Mutation LOF mutation Temperature Sensitive Mutation Screen for mutants with defects in cell cycle Preliminary 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 screen for conditional mutants Two Stages of the Cell Division Cycle CDC Screen 1 Primary screen 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 time 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 www ergito com cdc cell division cycle mutants great experiments 8 4 functions required at specific points in the cell cycle Budding Yeast CDC Mutants 50 cdc15 cdc28 cdc7 Terminal Phenotypes cdc9 cdc15 Arrest Point Asynchronous Population cdc15 terminal phenotype cell division cycle cdc mutants Defective in different steps of the cell cycle spindle pole body duplication initiation of DNA replication nuclear division cytokinesis cell separation OK so you ve got 50 cdc mutants How do you figure out which one is the most interesting one to study Budding Yeast CDC Mutants 50 cdc15 cdc28 cdc7 Terminal Phenotypes cdc9 Hartwell Defined Start Nutrients Start is a Critical Decision Point in G1 Start is a Branchpoint Leading to Alternate Fates Mating Pathway analysis DNA replication Wild type Budding SPB duplication DNA replication No DNA replication cdc7ts cdc31ts Budding cdc24ts No Budding SPB duplication SPB duplication DNA replication No DNA replication Budding No SPB duplication cdc28ts No Budding No SPB duplication In cdc28 mutant none of the downstream events occur Hartwell concluded that this was the key gene required for entry into the cell cycle Some CDC Mutant Phenotypes Should be 2 Spindle Poles CDC28 arrest point was closest to or at Start CDC28 is a Key Regulator of the Cell Cycle cdc28 Cdc28 is Required for Start Start is in G1 prior to the G1 S Transition Things to Remember We ll Return 1 MPF Humans Frogs Sea Urchins G2 M 2 Cyclin Frogs Sea Urchins G2 M 3 CDC28 S cerevisiae G1 S really mid G1 to late G1 Genetic analysis of the cell cycle in fission yeast S pombe Reproduces by fission Used to produce an African beer called pombe Pombe means beer in Swahili Like budding yeast grows rapidly well suited for classical and molecular genetic analysis Cells divide symmetrically cell size is very uniform at same position of cell cycle Grow in length not diameter easy to measure growth Sir Paul Nurse 2001 Nobel Prize Schizosaccharomyces pombe Recall from MCB 150 Evolutionary Distance Time from Last Common Ancestor Time from LCA Amount of Time Two Species Have Been Diverging From Each Other Evolution of Primates How far apart are S cerevisiae and S pombe Last Common Ancestor List Human and chimps 5 4 MYA 1 1 Human and mouse 91 MYA 2 Human and dog 92 MYA 1 Human and birds 310 MYA Human and frogs 360 MYA 15 Human and fish 450 36 Human vs fruit flies worms and mosquitos 990 MYA 46 S cerevisiae and S pombe 1 210 MYA 110 Human vs Fungi yeasts 1 520 MYA Human Animals vs plants 1 580 MYA 90 Evolution in Perspective S pombe cell cycle Genetic Nomenclature S cerevisiae S pombe Wild type CDC28 cdc2 Recessive LOF cdc28 cdc2 Dominant GOF CDC28 1 cdc2D Protein Cdc28 Cdc2 cdc mutants in S pombe Pollard and Earnshaw Fig 43 5 Nurse attempted to enrich for large cells fail to divide at restrictive temperature by sucrose gradient centrifugation At bottom of gradient found small cells instead These mutant cells wee1 divided at half normal size wee1 mutants Divide at normal size at permissive temperature but at half wild type size at restrictive temperature Wild type cells spend much of their time in G2 growing to critical size before entering mitosis wee mutants lose this G2 M growth control and divided at smaller size Nurse looked for more alleles of wee1 by screening for more mutants yielding small cells also found mutations in other genes that produced wee phenotype CDC Mutants in S pombe How did Nurse figure out which mutants were the most important ones to study Looked for genes in which dominant and recessive alleles GOF and LOF alleles gave opposite phenotypes Two key genes cdc2 and wee1 cdc2 Phenotypes LOF GOF Implication Cdc2 is