Test 3: Cell Cycle Flashcards
4 parts of Cell Cycle with identifiers & n
- G1: (1n): cell grows
- S: (>1n): DNA rep
- G2: (2) Prepares for mitosis
- M: (2n) Mitosis
Measuring Cell Cycle: 1. Determine Phase of the cell cycle is in. Check for S-phase by…
DNA replication only in S phase, so Thymedine-label (T in DNA) fed to cell for short period, washed, and look for cells with label
Measuring Cell Cycle: 1. Determine Phase of the cell cycle is in. Check for M-phase by…
Condensed chromosome
Measuring Cell Cycle: 1. Determine Phase of the cell cycle is in. Test for G1 by…
Cytometer, given it is only phase with 1 n cell. Label DNA with fluorescent probe.
Measuring Cell Cycle: 2. Measure length of each phase by…
% of cells in phase = % that phase takes up in cell cycle
Frog Oocyte Experiment: F. oocytes chosen because (4)
- large cell, easy to inject
- lots of cytoplasm, lots of pfoteins to be purified
- lots produced
- FROZEN IN G2 PHASE UNTIL SPERM ENTERS
Frog Oocyte Experiment: G2 phase frozen because either … (2)
- missing something needed to go thru cell cycle
2. something is inhibiting cell cycle
Frog Oocyte Experiment: To determine effect of G2 phase arrest, took oocyte and injected cytoplasm of cycling cells. Which phase of injected cell was effective? What was the conclusion?
M - oocyte starts mitosis. Hence, something is in M phase that triggers G2 -> M called Mitosis Promoting Factor (MPF)
Frog Oocyte Experiment: MPF exists in .. phase. It is highly…
M-phase only; conserved
Fission Yeast Experiment: Chosen because (4)
- Single Celled
- Easily Grown
- Haploid, hence easy genetics
- Only 2 cell cycle phenotypes
Fission Yeast Experiment: 2 cell cycle phenotypes were…
- arrested phenotype: won’t go through cell cycle and needs activator i.e. MPF
- wee phenotype: cycles too quickly, removed an inhibitor of sort, hence very small/no growth
Fission Yeast Experiment: Temperature-sensitive mutant has 2 phenotypes according to temperature. phenotype name and temperature corresponding to it?
- WT Phenotype /can cycle at permissive T
2. mut phenotype/wee or arrested at restrictive T
Fission Yeast Experiment: cdc2 is…. that unlike expected…
T-sensitive mut protein kinase that arrested at G2 like oocyte BUT did not react with MPF
Fission Yeast Experiment: cdc2 must be activated by…
cyclin
Fission Yeast Experiment: cyclin regulates kinase activity of cdc2 by forming… cdc2 is hence called…
cyclin:cdc2 complex; cyclin-dependent kinase
Fission Yeast Experiment: when cyclin:cdc2 complex is injected, oocyte…. Conclusion?
triggered m-phase. hence, the complex is GFP
Fission Yeast Experiment: level of cyclin v cdc2 across various cell cycles
cdc2 constant; cyclin peaks slowly to M and drops as soon as it ends, to rise again slowly from G1 to S to G2
cdc regulation: Upon further scrutiny, cdc2 + cyclin by itself does not wholly explain MPF activity. cak, wee1, and cdc25 is involved. explain.
G2: cak- & wee1-cdc2:cyclin:
cak phosphorylates cdc2 on Thr residue (act)
wee1 phosphoorylates cdc2 on Tyr residue (inhibit)
M: cdc25 removes phosphate from Tyr (act)
T/F cdc regulation: cdc25 is necessary for activation of GPF
T
cdc regulation: MPF has feedback loop that activates… which ….
activates cdc25, which deactivates effect of wee1 by dephosphorylating Tyr
cdc regulation: MPF’s sharp peaks of activity explained by…
self-propagation
cdc regulation: cak- mutation leads to
no cycling; G2
cdc regulation: wee1- mutation leads to
++ cycling; wee
cdc regulation: cdc25- mutation leads to
no cycling; G2
