Lecture 13 - Eukaryotic cell cycle control

Question 1

How is the eukaryotic cell cycle regulated.

Answer 1

• Cells utilize systems that respond to extra and intracellular signals/stimuli to control cell growth.
  
  • Integration of complex signalling pathways control when a cell divides
  • This orders system of biochemical switches is regulated and orchestrated by cyclin/CDK activities
  • Cyclin - CDKs ensure DNA replication occurs only once per cell cycle and that chromosomes are equally segregated into daughter cells

When defects arise in the cell division process, this can lead to diseases, such as cancer

Question 2

What are the phases of the eukaryotic cell cycle

Answer 2

G1 - Cell can either enter quiescence or enter the cell cycle
S - DNA Synthesis to form two identical copies of chromosome, organelles duplicate, centrosome duplicates
G2 - Check DNA is fully copied, centrosomes pulled to opposite poles, mitotic spindle formation
M - Mitosis, Chromosome condensation, nuclear envelope breakdown and chromosome segregation. Chromosome de-condensation and reformation of nuclear envelope, Cytokinesis. Two daughter cells.

Question 3

How were the factors that control cell growth identified?

Answer 3

The cell needs to establish co-ordination between different events occurring in space and time. The cell cycle ensures that the order of events is faithfully repeated in consecutive cell division cycles.

Identification of internal chemical signals that regulate cell division (Cell fusion experiments)
* Cell in M phase fused with cell in G2 phase causing mitosis in G2 phase cells. This meant could conclude that there is a mitosis promoting factor in M phase cells
* When the M cells were fused with G1, G2 or S the cell cycle can also be stimulated by M.
Rao & Johnson 1970
* There is an S-phase promoting factor in S-phase cells. Although there is an S-phase promoting factor only G1 cells respond to this factor.

Identification of proteins required for G2/M transition
Experimental System: Eggs and early embryos of African tree frogs (Xenopus) and marine invertebrates: sea urchin (Arbacia punctulata - Tim hunt)
Naturally synchronized i.e. arrested at specific point in the cell cycle. Undergo rapid synchronous cell divisions following fertilization
Therefore, it is possible to obtain cell extracts of biochemical analysis from specific points in the cell cycle.

Question 4

Describe xenopus oocyte maturation and the identification of maturation promoting factor.

Answer 4

1. Oocyte maturation in vitro
   a. G2 arrested oocyte - needs to be stimulated to become fertile
   b. Meiosis I (metaphase)
   c. Meiotic interphase
   d. Egg arrested in meiosis II (metaphase) - (Can go on to be fertilization by sperm and first cleavage)
2. Assay for MPF
   a. Egg arrested in metaphase II
   b. The cytoplasm is transferred to a G2 arrested oocyte
   c. MPF is applied and induces meiosis I metaphase
   d. Meiotic interphase and Meiosis II metaphase
   MPF activity rises and falls in parallel with mitosis.
   High MPF arrests the oocyte in meiosis until its abundance drops

Identification of “ cyclin” as a component of MPF
Autoradiograph showing newly synthesised protein. Sea Urchin oocytes fertilized and 35S Methionine added. Methionine is only incorporated within freshly produced protein. Protein extracts were taken and different time point postfertilization and analysed by SDS-PAGE.
Cyclin rises and falls soon after fertilisation.

Question 5

Why are frog oocytes a good model for idetnfication of gene encoding cyclin?

Answer 5

Frog oocytes provide an excellent model for identification of gene encoding cyclin. Addition of sperm chromatin to these extracts enabled mitosis to occur in the test tube. On addition of sperm chromatin the extracts are ablet to form mitotic spindles, condense the chromosomes, generate a metaphase plate and subsequently proceed through mitosis in vitro.

Question 6

How is cyclin B invovled in mitotic exit?

Answer 6

Cyclin B is a constituent of MPF
* Cyclin B levels parallel MPF activity
* Protein synthesis required (Protein deconstruction is also required)
* RNase treated extracts no longer undergo mitosis

Cyclin B mRNA
The mRNA has a ubiquitin ligase binding site which degrades cyclin B. Random truncation is used to remove sections.
Degradation of Cyclin B protein is required for mitotic exit. Addition of cyclin B mRNA reproduced a normal mitotic programme. However deletion of cyclin B5’ sequences produces a catalytically active truncated cyclin B protein. This shortened cyclin B construct is stable, as it cannot be degraded by the proteasome.
Its activity prevents mitotic exit
Therefore, cyclin B destruction is required for mitotic exit.