Genetics and Evolution Spring Term 2018 Describe and explain how mutagenesis screens can be used to find conditional mutations. Describe and explain how plasmid rescue can be used to identify key human genes. Discuss how genetics can be used to determine the role and function of genes. Evaluate how the yeast cell cycle exemplifies these learning objectives. Cell cycle genes - Leland Hartwell Nuclear migration G2 Spindle formation M Chromosome segregation nuclear division DNA replication S Bud emergence Spindle pole body duplication START G1 Growth Mutagenesis screen G1 S Cytokinesis Hartwell identified temperature sensitive mutants that do not progress through the cell cycle at the restrictive temperature. He did a mutagenesis screen to identify temperature sensitive mutants that didn't progress through the cell cycle. If you have a mutation in a cell cycle gene and you knock it out, the cells won't go through the cell cycle and die. Hartwell used budding yeast (reproduce by budding) as a model organism. He used budding yeast because it's a single cell eukaryote that can be grown on an agar plate. You can see clearly the cell cycle stages by the cell morphology. The cells are easily recognisable under the microscope. G2 M wild-type mutant Temperature sensitive mutants have a wild-type phenotype at permissive conditions and are 'conditional'. This means that you can reveal the mutant phenotype but in other conditions you can rescue the phenotype and maintain the living organism. Without the conditional feature you would lose the mutation as it would be lethal. The screen looks for mutants identical to wild types in permissive conditions - you can rescue the mutant at a permissive temperature. G1 S G2 M wild-type mutant All cell division cycle (cdc) mutants arrest at a specific point in the cell cycle. At the restrictive temperature mutations in genes required for progression from the G1 phase cause yeast to arrest at the end of the G1 mutant Restrictive conditions - different temperatures. At one temperature they have a wild type phenotype and a mutant one at a different temperature. If the gene is knocked out, the phenotype tells you the function of the gene. This gene is required for the transition from G1 to S phase. mutant Normally it would be lethal and the cell would die but the permissive conditions allowed them to grow at a certain temperature. Key feature of temp sensitive mutations: conditional, cell division cycle mutants - they arrest at a specific point in the cell cycle. 1
G1 S G2 M From these screens, Hartwell identified all the key cell cycle regulator genes. wild-type G2 phase cdc mutant If you have a culture of a particular cell cycle mutant in restrictive conditions, the cells will arrest at the same point in the cell cycle. M phase cdc mutant S phase cdc mutant All cdc mutants a specific phenotype: Some cdc Mutants Wild type cdc 8 G1 S G2 M wild-type - cdc 24 cdc 10 non cdc mutant A non cell cycle