L14: The genetics of cell cycle control and regulation of mitotic entry Flashcards
1
Q
Budding yeast cell cycle (vs Euk cell)
A
- Main 4 phases of cell cycle are conserved across organisms
- Major control point at the G1/S transition: START (called restriction point in Euk)
- Yeast cells tend to change cell morphology more profoundly than Euk
- Bud is present on cell at all stages but G1 - mother cell is larger component, bud becomes daughter
2
Q
Lee Hartwell Expts
A
- Used selective temperatures to identify genes that control cell division
- Isolated a library of temperature sensitive mutants in S.cerevisiae
- To identify which genes were mutated he used a process of complementation (adding back functional copy of mutated gene -> rescue phenotype at non-permissive temps.)
- A wild type yeast genome was digested and ligated into plasmids - cloning genes by complementation
3
Q
Genetic complementation (Hartwell - results)
A
- cdc28 temp. sensitive cells for low temperatures
- No colony mutation at 35 degrees with other genes
- Transformed cells w/ plasmid library of wt cdc28 plasmids
- Phenotype rescued in mutant transformed with cdc28 plasmid
- Temperature sensitive mutants affect protein stability and function
- Some cdc28Ts mutants arrest in G1 phase; Cdc28 is a component of SPF (Cdk)
-> Gene complementation to identify compensatory mechanisms identified in G1 cyclins
-> 9 different Cdks identified through cdc28; cell cycle stage specific
4
Q
G1 cyclin in wt vs cdc28Ts
A
- Kinase domain of Cdk has weakened BS for cyclin in Ts mutant; normally, the two remain bound at any temp, but low affinity in mutant, dissociates at high temperatures
5
Q
Alternate mechanism for rescuing ts mutant
A
- Dosage suppression allows growth at non-permissive temperatures
- cdc28Ts cells transformed w/ high-copy G1 cyclin plasmid
- Equilibrium shifted, colonies can form at high temperature
6
Q
Fission yeast intro (cell cycle, key ps of experiments)
A
- S.pombe
- Start as small rod-shaped cells; length of cell increases throughout cycle - able to determine stage by length
- Identification of Cdc mutants in S.pombe identified a G2/M arrest
- Gene complementation developed to identify cell cycle regulators
- S.cerevisiae gene library used
7
Q
cdc2Ts in S.pombe (Nurse lab)
A
- cdc2Ts transformed w/ cdc28 (homolog from budding yeast), restore function at high temp/35 degrees
-> Cdc28 homologues are conserved in yeast species
8
Q
Testing conservation in eukaryotes (5 step process) i.e designing primers to clone genes from related organisms
A
- AA seq means you can predict potential DNA seq. of coding region
- Synthesise degenerate oligo-nt corresponding to DNA seq. coding for regions 1 and 2
- Use oligo-nt to amplify DNA btwn regions 1 and 2 and then the whole open reading frame from cDNA source
- Clone open reading frame into yeast expression plasmid
- Test conservation by complementation
-> Cdks are universally conserved in Euk.
9
Q
Controlling the G2/M transition (cdc mutants)
A
- cdc2+; optimal cell division
- cdc2-; unable to enter mitosis, cell growth continues
- cdcD; constitutively active, enter mitosis inappropriately
, cell growth insufficient (‘wee’ protein)
-> cdc2+ is both required and rate limiting for mitosis
10
Q
G2/M transition and phosph.
A
- Mitotic cyclin expression occurs in late S/G2 phase
- MPF remains inactive due to inhibitory phosph. - Wee1
- Cdc2 requires phosph. at T161 for activity (Cdk activating kinase/CAK - fully activates, increasing catalytic activity, unfolds T-loop)
- Inhibitory phosph. at Y15 must be removed for full activity
- Cdc25 removes inhibitory phosph. to induce mitosis at G2/M transition
- Process occurs due to a number of feedback loops
11
Q
Temporal regulation of mitosis by feedback loops
A
- Wee1 inhibits Cdk via inhibitory phosphorylation
- Cdc25 activates Cdk
- Cdk enhances Cdc25
- Cdk inactivates Wee1
-> enables rapid transition towards mitosis
-> opposing and balanced CDK and Wee1 activities ensure regulated mitosis
