Microbiology 21: (Fry) How cdks were identified

Question 1

Why use yeast in eukaryotic genetic study?

Answer 1

Simple unicellular eukaryotic organism that doubles every few hours

Cell cycles very similar to higher eukaryotes

Yeast lab strains are usually haploid -> ideal for loss of function mutagenesis screens

Question 2

What is a conditional lethal mutant? Example?

Answer 2

A gene that results in death of an organism when placed in certain conditions

e.g. a mutant protein works fine at lower temperature (permissive), unstable at higher temperature (restrictive)

Question 3

Cdc2, Cdc 25 and Wee1 - why are these yeast mutants so significant?

Answer 3

Cdc2 (kinase) - Cells grow abnormally long, cant divide
Cdc25 (phosphatase) - Cells grow abnormally long, cant divide
-> therefore activator of G2/M transition as mutation prevents mitotic entry

Wee1 (kinase) - Cells much smaller than usual, divide too quickly
-> therefore inhibitor of G2/M transition as mutation leads to premature mitotic entry

Question 4

Explain the process of gene cloning by complementation - use Cdc2 as example

Answer 4

Temperature sensitive Cdc2 mutant yeast strain cultured
- Cells arrested in 1 stage of cell cycle (G2)

Genes from yeast genomic library were transformed into yeast until 1 was found that rescued the mutant phenotype at the restrictive temperature
-> this identified gene function

Same process was repeated with human genes -> human Cdc2 gene was found to also rescue phenotype -> same proteins involved

Cdc2 first Cdk to be identified

Question 5

Advantages to using marine invertebrate oocytes, eggs and embryos in cell cycle research?

Answer 5

Easily available in large quantities

Synchronous divisions so extracts represent defined cell cycle stages

Large enough to be injected with proteins, DNA extracts etc.

Optically clear enough to visualise what is going on inside cell by light microscopy