Professor Fry's Work

Question 1

How long is the average cell cycle?

Answer 1

24 hours

Question 2

What happens in early prophase?

Answer 2

Chromosome condensation

Question 3

What happens in late prophase?

Answer 3

Centrosomes move to opposite ends of the cell

Question 4

What happens in pro-metaphase?

Answer 4

Nuclear envelope breaks down

Mitotic spindle assembles

Question 5

What happens in metaphase?

Answer 5

Chromosomes align at metaphase plate

Full bipolar attachment to spindle

Question 6

What happens in telophase?

Answer 6

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

Question 7

What are the two types of mitotic spindle?

Answer 7

Interpolar

Kinetochore

Question 8

What happens in Anaphase A?

Answer 8

Sudden loss of cohesion between the sister chromatids

Question 9

What happens in Anaphase B?

Answer 9

Whole spindle elongates increasing full separation

Question 10

What happens in cytokinesis?

Answer 10

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

Question 11

What is the difference between somatic and embryonic growth?

Answer 11

In somatic cells there is growth between divisions and a few origins of replication.
Embryonic cells divide synchronously

Question 12

Outline Rao and Johnson’s cell fusion experiment

Answer 12

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

Question 13

What is cyclin abundance controlled by?

Answer 13

Regulation of transcription
Regulation of translation during oogenesis
Regulation of destruction

Question 14

How were yeast used to identify cdks?

Answer 14

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

Question 15

What are the cdc’s in fission yeast?

Answer 15

cdc2-cdc13 - G2–>M

cdc2-cig2 - Start point

Question 16

What are the cdc’s in bussing yeast?

Answer 16

CDC28-Clb1-4 - G2->M

CDC28 - Cln1-3 - Start point

Question 17

How is oocyte maturation triggered in Xenopus bivis?

Answer 17

The injection of progesterone into the cytoplasm inhibits AC which was stimulating PKA which was inhibiting the G2->M transition

Question 18

Outline the maturation assay

Answer 18

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

Question 19

What did Maller and Gehardt do?

Answer 19

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

Question 20

Who identified cyclins and how?

Answer 20

Tim Hunt using radioactive amino acids

Question 21

How are cdks regulated?

Answer 21

By cyclin binding
Phosphorylation
Degradation of cyclins
Small Protein Binding

Question 22

How are cdks regulated by cyclin binding?

Answer 22

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.

Question 23

How does phosphorylation regulate cdks?

Answer 23

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

Question 24

How are cyclins degraded?

Answer 24

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.

Question 25

How do small proteins regulate cdks?

Answer 25

P16 stops cyclins binding to Cdk4 and Cdk6

P21 and p27 inactivate any cyclin complex containing cdk2

Question 26

What are the 4 major cell cycle checkpoints?

Answer 26

Restriction
DNA Damage
DNA replicative Stress
Mitotic Spindle

Question 27

What must every checkpoint have?

Answer 27

Sensor, Signalling mechanism and effector

Question 28

Outline the restriction point checkpoint

Answer 28

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

Question 29

Outline the DNA damage checkpoint

Answer 29

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

Question 30

Outline the DNA replication stress checkpoint

Answer 30

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

Question 31

Outline the mitotic spindle checkpoint

Answer 31

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