Professor Fry's Work Flashcards
How long is the average cell cycle?
24 hours
What happens in early prophase?
Chromosome condensation
What happens in late prophase?
Centrosomes move to opposite ends of the cell
What happens in pro-metaphase?
Nuclear envelope breaks down
Mitotic spindle assembles
What happens in metaphase?
Chromosomes align at metaphase plate
Full bipolar attachment to spindle
What happens in telophase?
Disassembly of the spindle
Reformation of the nuclear envelope
Decondensation of the chromosomes
What are the two types of mitotic spindle?
Interpolar
Kinetochore
What happens in Anaphase A?
Sudden loss of cohesion between the sister chromatids
What happens in Anaphase B?
Whole spindle elongates increasing full separation
What happens in cytokinesis?
Late Anapahase - Actomyosin contractile ring assembles
Telophase - Begins to contract and additional plasma membrane is deposited creating a visible cleavage furrow
Cytokinesis - Constriction and abcission
What is the difference between somatic and embryonic growth?
In somatic cells there is growth between divisions and a few origins of replication.
Embryonic cells divide synchronously
Outline Rao and Johnson’s cell fusion experiment
Fused human HeLa cells to create heterokaryons.
Mitotic + G1 -> Condensation
S + M, M+ G2 -> Induced mitosis
G1 +S -> S phase
G2 + S -> continued as normal
G1 + G2 -> continued as normal
These showed the existence of an MPF and SPF
What is cyclin abundance controlled by?
Regulation of transcription
Regulation of translation during oogenesis
Regulation of destruction
How were yeast used to identify cdks?
When fission yeast do not undergo mitosis they just continue to elongate. Temperature sensitive mutants were created, cdc2, cdc 25, cdc 13 showed no mitosis and wee1 showed early mitotic entry. A combination of cdc 25 and wee1 had a WT phenotype showing these mutations were competitive.
These then underwent a complementation analysis which identified the genes responsible for these random mutations.
The mutations were then tested by overexpression in wild-type cells which led to correction in wee1 and cdc 25 however cdc 2 and cdc 13 remained long as cdc 25 is a RLE
What are the cdc’s in fission yeast?
cdc2-cdc13 - G2–>M
cdc2-cig2 - Start point
What are the cdc’s in bussing yeast?
CDC28-Clb1-4 - G2->M
CDC28 - Cln1-3 - Start point
How is oocyte maturation triggered in Xenopus bivis?
The injection of progesterone into the cytoplasm inhibits AC which was stimulating PKA which was inhibiting the G2->M transition
Outline the maturation assay
Cytoplasm from M-phase cell into oocyte triggered M phase, this was repeated to ensure it wasn’t excess progesterone from the cell membrane.
Cytoplasm from an interphase cell was injected into the oocyte which remained arrested in G2
What did Maller and Gehardt do?
Repeated the maturation assay and found a burst of phosphorylation upon oocyte maturation
Who identified cyclins and how?
Tim Hunt using radioactive amino acids
How are cdks regulated?
By cyclin binding
Phosphorylation
Degradation of cyclins
Small Protein Binding
How are cdks regulated by cyclin binding?
In the unbound state the T-loop blocks the active site and PSTAIRE is far from the active site. When cyclin binds it pushes PSTAIRE towards the active site allowing a salt bridge to form between E (glutamate) and lysine 33. It also causes uncoiling of the alphaL12 helix which pulls the T-loop away from the active site.
How does phosphorylation regulate cdks?
Wee1 and CAK phosphorylate cdk-cyclin complex simultaneously.
Wee1 adds two inhibitory phosphates at Thr-14/Tyr-15
CAK adds a stimulatory phosphate at Thr-161
When the cyclin-cdk complex is needed it is activated by cdc25 (a phosphatase) removing the inhibitory phosphates
How are cyclins degraded?
E3 aka the Anaphase Promoting Complex/Cyclosome detects the D box, takes a Ub off E2 and sticks it on the protein. When polyubiquitinated the lid of the proteasome recognises it, unfolds it and feeds it into the lumen where the protein is degraded.
