Chang, F. & I. Herskowitz (1990) Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2. Cell 63: 9991011.
Prior to this report, all the mutants isolated based on their resistance to a-factor arrest were also found to be sterile and did not induce FUS1 or other genes regulated by the mating-type pheromone response pathway. In this article the authors search for mutants that separate these phenotypes. That is, they searched for mutants that were resistant to pheromone arrest but were still capable of signaling.
To understand the mechanism of cell cycle arrest by the mating-type pheromones, the reader must become familiar with the regulatory controls of the cell division cycle, particularly those controlling the G1 to S transition. The description in a cell biology text should be sufficient. In Saccharomyces, CDC28 encodes the p34Cdc2 kinase homologue that is the key regulator of both the G1 to S and G2 to M transitions. Cdc28 protein is a cyclin-dependent kinase and is activated by binding with a cyclin protein. Saccharomyces has two classes of cyclins used to regulate the cell cycle encoded by the CLN and CLB genes. CLN1, CLN2, and CLN3 encode the G1 cyclins. That is, they are expressed during the G1 phase, although their expression patterns are distinct. The Cln proteins exhibit sequence homology but are not identical, and binding of any one of these to Cdc28 is sufficient to traverse START. Three different cyclins, encoded by the CLB genes, regulate Cdc28 during the G2 to M transition and are referred to as the G2 cyclins. A culture of unsyn-chronized cells when treated with pheromone will proceed through S, G2, and M and arrest in G1 at START. In earlier studies Hereford & Hartwell (1974) demonstrated that a-factor arrested cells at START and this was coincident with the requirement for Cdc28 activity. Thus, the G1 cyclins are the likely targets of a-factor arrest. For these reasons, Chang & Herskowitz investigated the CLN genes as targets of FAR1 -mediated cell cycle arrest in response to a-factor.
1. Discuss the details of the mutant isolation screen that enabled the authors to isolate this novel class of mutants resistance to a-factor arrest. How are they novel?
2. Describe the following phenotypes of farl mutants in the presence of a-factor.
(a) Changes in the cell cycle.
(b) Colony formation.
(c) Transcription of downstream target genes like FUS1 and agglutinin.
(d) Morphological changes.
(e) Mating competency.
3. What results indicate that Farlp acts only in the a-factor response pathway that regulates cell division and not in other pathways regulating the cell cycle, such as those that respond to changes in nutrient levels?
4. Summarize the evidence that FAR1 expression is regulated by pheromone via the pheromone response pathway.
5. Based on the results shown in Figure 6, the authors conclude that Farlp is a negative regulator of Cln2p and not Clnlp or Cln3p. These conclusions are summarized in Figure 9.
(a) What evidence indicates that all three Cln proteins are inhibited in MATa cells exposed to a-factor?
(b) Which mutant allele is epistatic, cln2 or farll Which gene is downstream?
(c) What evidence indicates that Farlp is a negative regulator of Cln2p? (Remember that Cln2p is a positive regulator of the G1 to S transition.)
(d) What evidence indicates that clnl A and cln3A are not downstream of farl!
6. Discuss the reasons why the authors suggest that the Farl protein has additional roles in mating other than its role in G1 arrest.
7. Why is it necessary to use the farl A null mutation and not a farl point mutation to conclude that Farlp has no role in the mating-type pheromone response pathway itself?
8. Describe how you would select/screen for mutants in the a-factor dependent inhibitor of Clnlp, i.e. the one referred to as X in Figure 9. Be specific with regard to the genotype of the starting strain.
Hereford, L.M. & L.H. Hartwell (1974) Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. J. Mol. Biol. 84: 445-461.
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