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Flashcards in Professor Fry's Work Deck (31):
1

How long is the average cell cycle?

24 hours

2

What happens in early prophase?

Chromosome condensation

3

What happens in late prophase?

Centrosomes move to opposite ends of the cell

4

What happens in pro-metaphase?

Nuclear envelope breaks down
Mitotic spindle assembles

5

What happens in metaphase?

Chromosomes align at metaphase plate
Full bipolar attachment to spindle

6

What happens in telophase?

Disassembly of the spindle
Reformation of the nuclear envelope
Decondensation of the chromosomes

7

What are the two types of mitotic spindle?

Interpolar
Kinetochore

8

What happens in Anaphase A?

Sudden loss of cohesion between the sister chromatids

9

What happens in Anaphase B?

Whole spindle elongates increasing full separation

10

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

11

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

12

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

13

What is cyclin abundance controlled by?

Regulation of transcription
Regulation of translation during oogenesis
Regulation of destruction

14

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

15

What are the cdc's in fission yeast?

cdc2-cdc13 - G2-->M
cdc2-cig2 - Start point

16

What are the cdc's in bussing yeast?

CDC28-Clb1-4 - G2->M
CDC28 - Cln1-3 - Start point

17

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

18

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

19

What did Maller and Gehardt do?

Repeated the maturation assay and found a burst of phosphorylation upon oocyte maturation

20

Who identified cyclins and how?

Tim Hunt using radioactive amino acids

21

How are cdks regulated?

By cyclin binding
Phosphorylation
Degradation of cyclins
Small Protein Binding

22

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.

23

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

24

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.

25

How do small proteins regulate cdks?

P16 stops cyclins binding to Cdk4 and Cdk6
P21 and p27 inactivate any cyclin complex containing cdk2

26

What are the 4 major cell cycle checkpoints?

Restriction
DNA Damage
DNA replicative Stress
Mitotic Spindle

27

What must every checkpoint have?

Sensor, Signalling mechanism and effector

28

Outline the restriction point checkpoint

Sensor - Decrease in stimulatory GFs/Increase in inhibitory, indicators of bad growth conditions
Signalling - Down regulation of MAPK, PI3K etc
Effector = Decrease in cyclin D, Increase in p16, increase in p27

29

Outline the DNA damage checkpoint

Sensor - Proteins that recognise DNA error
Signalling - Multiple protein kinases that cause a DNA damage signal
Effector - Stabilises p53 leading to an increase in p21, inactivation of cdc25, expression of genes that promote apoptosis

30

Outline the DNA replication stress checkpoint

Sensor - stalled replication, ss DNA
Signalling - Protein kinases especially ATR
Effector - Inactivation of cdc 25 and stabilisation of p53

31

Outline the mitotic spindle checkpoint

Sensor - Attachment -proteins on kinetochores, OR/AND Tension between sister chromatids
Signalling - Protein Kinases
Effector - Prevent destruction of cyclins which would lead to mitotic exit, inhibits APC/C