Regulation of the Cell Cycle

Question 1

Cell cycle events must be regulated so that…

Answer 1

1. Events occur in the correct sequence with the correct timing
2. Cell proliferation is appropriate (only occurs with sufficient nutrients and correct cell-cell signaling)
3. Cell cycle is coordinated with cell growth (mass of cell doubles every cell cycle, so average cell mass stays constant)

Question 2

What conclusion can be reached from cell fusion experiments? (Where 2 cells in different phases are fused)

Answer 2

Dominant factors in cytoplasm can drive cells into later stages of mitosis.

Question 3

Why are sperm and egg cells valuable in researching the cell cycle?

Answer 3

It is possible to synchronise the fertilisation of eggs, so they will all be in the same stage of mitosis.

Question 4

How can the existence of a maturation promoting factor be demonstrated

Answer 4

Cytoplasm extracted from mature egg can induce maturity without fertilisation- there must be a maturation promoting factor (MPF) present.

Question 5

What is the reduction of MPF activity associated with?

Answer 5

Ca2+induced cyclin degradation

Question 6

Outline the stages in the activation of the first cell cycle after fertilization

Answer 6

1. The egg nucleus is arrested at metaphase of meiosis II
2. Levels of MPF normally decrease at this stage of the cycle
3. Levels of MPF instead remain high
4. Activation of Cam-kinase II after fertilization triggers
   degradation of cyclin
5. Meiosis II completes
6. The male and female pro-nuclei fuse
7. The zygote enters mitosis
8. Embryonic cell cycles omit G1 and G2 for speed

Question 7

How was MPF identified as composed of a Cyclin B/Cyclin dependent kinase (Cdk) complex?

Answer 7

Use of temperature sensitive mutants (cells which only undergo mitosis at low temperatures)

Question 8

Explain the how temperature dependent mutants were used.

Answer 8

1. Break up genome of wild-type into fragments with restriction enzymes and use plasmids to transform these into mutants.
2. Those mutants which have plasmids containing the functioning form of the gene will survive and continue to divide at restrictive temperature.
3. Plasmid can then be isolated.

Question 9

How does cyclin B accumulation and degradation regulate mitosis?

Answer 9

1. Onset of mitosis is triggered by increase in CDK activity - this happens in part by increase in cyclin B concentration.
2. Exit from mitosis involves proteolysis of cyclin B and thus inactivation of CDK

Question 10

Why is the beta-barrel surrounding the fluorophore of GFP advantageous?

Answer 10

It protects cells from the fluorophore, allowing GFP to be used in living tissue.

Question 11

MPF isn’t the only cyclin/Cdk involved cell cycle regulation…

How does the regulation of the cell cycle by cyclin/Cdk in yeasts and mammalian cells differ?

Answer 11

In yeasts, there is a single Cdk, but multiple cyclins which bind at different stages of the cycle.

In mammalian cells, there are multiple Cdks and multiple cyclins

Question 12

How does Cdk coordinate the cell cycle

Answer 12

Phosphorylation of multiple targets throughout the cell.

Question 13

What two steps are required to fully activate a Cdk/

Answer 13

1. Cyclin binding- partially activates and exposes T-loop.

2. Phosphorylation of T-loop.

Question 14

How else is Cdk activity regulated?

Answer 14

1. Phosphorylation of an inhibitory site by Wee1 kinase

2. Cdk inhibitor proteins (CKI)- activated by events such as DNA damage to prevent replication of damaged cells.