D103 - Lecture 23 - Cell Cycle III.docx

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Biological Sciences
Grant Mac Gregor

A. Clickers a. Mutation that would produce a protein with a temperature sensitive mutation i. (B) missense mutation b. Change the Thr to His i. Positive charge ii. Can't put a phosphate on the threonine (GAS, phosphorylated by CAK) iii. Leading to large cells B. Budding versus Fission a. Budding i. Studying the G1 to S phase transition b. Fusion i. Studying G2 to M transition B. Yeast a. Good model system b. Small genome size c. Able to proliferate in a haploid state i. Makes it easy to isolate mutants ii. Doesn't require creation of a homozygous recessive B. Temperature Sensitive (ts) a. Conditional mutant b. Only see the mutation effect under a certain temperature c. Inactive - restrictive temperature d. Active - permissive temperature e. Can be cold-sensitive or heat-sensitive B. Process of temperature sensitive yeast a. Incubate for a period of time after mutation i. Add mutagen to yeast liquid culture ii. Allow the mutation to become fixed in the genome iii. Plate the mutated yeast onto solid media iv. Colonies will grow (each from a single yeast cell) v. Velvet transfer (diagram) - replicate plating 1. Velvet plate - 2 plates a. At permissive temp b. At restrictive temp i. Look for missing colonies ii. Compare to permissive temp plate b. Propagate the missing colony from the permissive plate B. Isolation of cell cycle mutants a. Premature entry i. Smaller ce
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