Lecture 14 - Genetics of cell cycle control

Question 1

Describe the budding yeast (saccharomyces cerevisae) cell cycle.

Answer 1

1. The parent and daughter cell of S. Cerevisiae are different sizes. During G1 the growth phase the daughter cell grows to a normal size.
   
   2. The major G1/S transition control point is START
   3. Spindle pole body duplication occurs and a bud begins to emerge
   4. DNA is replicated and the bud continues to grow
   5. In the G2 phase spindle forms and the nucleus migrates to the midpoint of the mother and daughter cells
   6. Chromosome segregation and nuclear division occurs
      Cytokinesis takes place forming one parent cell and a smaller daughter cell

Question 2

How were genes required for cell cycle progression identified in s. cerevisae

Answer 2

Lee Hartwell used selective temperatures to identify the genes that control cell division. By 1967 he had isolated approximately 400 different temperature sensitive mutants in S. Cerevisiae. The temperature sensitive (Ts) mutants would grow at 25 ̊C but not at 35 ̊C. To identify which genes were the ones that had been mutated he used a process of complementation (add/block functional gene). Addition of the functional copy of the gene rescues cells at non-permissive temperatures.
A wild type yeast genome was digested and ligated into plasmids - cloning genes by complementation.

The cdc28ts yeast strains, carrying a temperature-sensitive mutation in the CDC28 gene, were cultivated at 35°C. Following transformation with a plasmid library containing wild-type Saccharomyces cerevisiae DNA, the inability of the cells to form colonies at the non-permissive temperature signified that the introduced DNA was not complementary to the mutated gene. Conversely, the formation of colonies indicated successful complementation, suggesting that the transformed DNA could compensate for the loss of the CDC28 gene function. Subsequently, plasmids were isolated from the colonies, enabling the identification and further analysis of the specific genes responsible for the complementation of the cdc28 mutation.

Cdc28, a cyclin-dependent kinase in budding yeast, is integral to cell cycle regulation. Temperature-sensitive cdc28 mutants revealed that Cdc28 is crucial for G1 phase progression, implicating it in the S-phase promoting factor. Through gene complementation studies, compensatory mechanisms were identified, highlighting the involvement of G1 cyclins in collaboration with Cdc28 for the G1-to-S phase transition.

Question 3

What mechanisms contribute to the ability of wild-type cells to form colonies at both normal and high temperatures, compared to Cdc28 temperature-sensitive mutant cells, and how does dosage suppression play a role in overcoming this defect?

Answer 3

Wild type cells formed colonies at both normal and high temperatures as Cdc28 and the G1 cyclin could bind to form SPF.

Cdc28ts (temperature sensitive mutant) cells didn’t from colonies and the cell cycle was arrested in G1. This is because the temperature sensitive cells had low affinity cdc28 for the cyclin in comparison to the wild type Cdc28.

Dosage suppression allows growth at non-permissive temperatures. (Dosage suppression is a genetic phenomenon where an increase in the copy number of a particular gene compensates for a mutation in another gene). In Cdc28ts cells transformed with high copy of G1 cyclin plasmid, SPF and colonies would both form at both temperatures as the excess of G1 cyclin would shift the equilibrium.

Gene complementation identified several cyclins. Cyclin expression is cell cycle stage specific.

Question 4

What methods were used to identify cell cycle regulators in S. pombe, and how did gene complementation with a S. cerevisiae gene library aid in this process?

Answer 4

During the S. pombe cell cycle the length of the yeast increases which is used to identify the stage of the cell cycle a cell is in.
Identification of Cdc mutants in S .pombe identified G2/M arrest. Gene complementation was developed to identify cell cycle regulators.
Gene complementation was performed using a S. cerevisiae gene library and researchers hoped for conserved function between yeast species.

They found that Cdc28 rescues S. pombe cdc2ts mutants at non permissive temperatures, The Cdc28 is the same as in S. cerevisiae. This showed that Cdc28 homologues are conserved in yeast species and lead to the question as to whether they were conserved in eukaryotes.

Question 5

What experimental strategy was employed to demonstrate the structural and functional conservation of CDK1 across eukaryotes?

Answer 5

Cdk1 is structurally and functionally conserved in eukaryotes
1. Amino acid sequence means you can predict the potential DNA sequence of coding region
2. Synthesize degenerate oligonucleotides corresponding to the DNA sequence coding for regions 1 and 2 which showed high conservation
3. Use oligonucleotides to amplify DNA between regions 1 and 2 and then the whole open reading frame from cDNA source
4. Clone open reading frame into yeast expression plasmid
5. Test conservation by complementation
CDK1 was able to compensate for S. pombe cdc2Ts
Therefore Cdc28 = Cdc2 = CDK1

Question 6

How do cdc mutants shed light on the key roles of CDK regulation in controlling the G2/M transition in the cell cycle, and what additional regulatory networks beyond MPF formation are involved in ensuring proper mitotic progression?

Answer 6

Controlling the G2/M transition (cdc mutants)
* Wild type cdc2+ caused optimal cell division
* Cdc2- meant the cell was unable to enter mitosis and cell growth continues
* CdcD meant that the enzyme was constitutively (always) active so the cell would enter mitosis inappropriately and the cell growth was insufficient (wee)
Cdc2+ is both required and rate limiting for mitosis. It is regulated to prevent inappropriate mitosis.

Phosphorylation events aligned to cell cycle progression
CDK are regulated by cyclin binding. Mitotic CDK activity is regulated by activating and inhibitory phosphorylation in addition to cyclin binding.
Formation of MPF is not sufficient to control mitosis. This requires additional regulatory networks : wee-1-kinase and cdc25 phosphatase.

Question 7

How is the G2/M transition regulated in s. pombe?

Answer 7

G2/M transition is regulated by the phosphorylation state of MPF
Mitotic cyclin expression occurs in late S/G2 phase.
MPF remains in active due to inhibitory phosphorylation - wee 1
Cdc2 requires phosphorylation at T161 for activity - CAK (CDK Activating Kinase)
Inhibitory phosphorylation at Y15 must be removed for full activity
Cdc25 (protein phosphatase) removes inhibitory phosphorylation to induce mitosis at G2/M transition

Activation of MPF is controlled by Wee1 and Cdc25
* Mitotic CDKs are inactive in G2 phase due to inhibitory phosphorylation mediated by Wee1
* Removal of phosphorylation by Cdc25 phosphatase enables rapid transition into mitosis

Temporal regulation of mitosis is controlled by rapid feedback loops:
1. Wee1 inhibits CDK via inhibitory phosphorylation
2. Cdc25 activates CDK by removing phosphates
3. CDK enhances Cdc25 activity and inactivates Wee1 causing rapid full activation

Opposing Wee1 and cdc25 activities regulate CDK kinase activation
* Deficit of Cdc25 or excess of Wee 1 prevents mitosis leading to elongated cells
* Excess of Cdc25 or deficit of Wee1 leads to small cells due to inappropriate mitosis
Opposing and balanced CDK and Wee1 activities ensure regulated mitosis