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
cdc regulation: wee1- and cdc25- mutation leads to…
++ cycling; wee
MPF: is a kinase. it effects 4 things
- chromosome condensation
- nuclear envelope breakdown
- mitotic spindle formation
- cyclin degradation
MPF: 1. chromosome condensation… by
phosphorylating histones and condensins
MPF: 2. nuclear envelope breakdown… by
phosphorylating lamin (which nuclear envelope is made of to prevent protein protein interaction
MPF: 3. mitotic spindle formation… by
dynamic instability changed by +++ ++ MAPs associated with microtubules; phosphorylates MAPS
MPF: 4. cyclin degradation… by
end of mitosis to decrease cyclin steeply
Cyclin:CDK is common mode of regulation for cell cycle. … ocntrols entry into S-phase from G1
cdc2:G1 cyclin
Cyclin:CDK: G1 -> S entry point critical b/c
Chromosome duplicates in S-phase, commits to mitosis after that
G1 Cyclin:Cdc2: cyclin substrate is…
Rb, which inhibits transcription by binding to ts E2F
G1 Cyclin:Cdc2: 1 cyclin, Rb, E2F. Relate.
Rb:E2F prevents transcription.
G1:cc2 phosphorylates Rb
E2F now free for transcription of genes for S-phase
Cyclin:CDK: S-phase itself trigged by…
S-Cyclin:Cdc6
S-Cyclin:Cdc6: S-cyclin… (2)
- initiates DNA replication
2. Prevents re-replication of DNA
S-Cyclin:Cdc6: Relate cdc6, Origin Recognition Complex (ORC), ori, and S-Cdk
- ORC is bound to ori
- cdc6 binds to ORC to recreate pre-replicative complex
- S-cdk triggers phosphorylates cdc-6 & triggers S-phase
S-Cyclin:Cdc6: S-cyclin initiates DNA replication by…
@ ori, pre-replication complex sits on DNA and waits for S-phase. s-Cdk triggers replication as cdc6 is phosphorylated and comes off of ORC
S-Cyclin:Cdc6: S-cyclin prevents rereplication by…
s-Cdk phosphorylates cdc6, which is soon degraded. cdc6-P triggers its own destruction so it does not form more pre-replicative complex
DNA Damage: when damaged, cell…
cell arrests to give time for repair
repair
DNA Damage: relate p53 and p21
DNA damage leads to + transcription of p53 for DNA repair
leads to + transcription of p21 for cdk inhibition
