Article 15

Valtz, N., M. Peter, & I. Herskowitz (1995) FAR1 is required for oriented polarization of yeast cells in response to mating pherones. J. Cell Biol. 131: 863-873.

Chenevert et al. (1994) devised a screen to identify mutants defective in the ability to undergo the properly oriented morphological changes (schmoo formation) associated with pheromone exposure. They reasoned that such mutants would be capable of mating to a wild-type strain but should exhibit defects when paired with an enfeebled mating partner. Among the several mutant genes identified, Chenevert et al. (1994) identified new alleles of FAR1 that they called farls alleles. Article 15 characterizes these farls alleles.

Chang & Herskowitz (Article 8) identified FAR! and characterized its role in cell cycle arrest. They demonstrated that Farlp functions as a negative regulator of the G1 cyclin Cln2p. This inhibitory effect involves the direct binding of Farlp to Cdc28p/Cln2p cylin-dependent kinase but, in contrast to other cyclin kinase inhibitors, Farlp binding does not appear to result in the inhibition of kinase activity and thus the mechanism of action is novel (Peter & Herkowitz, 1994; Gartner et al., 1998). Article 15 focuses on quite a different function of Farlp, namely cell polarization during schmoo formation.

Chang & Herskowitz (Article 8) suggested a role for Farlp in cell polarization during mating based on their initial analysis of different farl mutant phenotypes. Additionally, Chang (1991) describes farl-c, a C-terminal truncation mutation, capable of cell cycle arrest in response to pheromone but defective in mating, perhaps due to an inability to orient toward the mating partner. These studies illustrate the value of detailed analysis of the pleiotropic phenotypes of multiple mutant alleles.

1. Describe the pheromone confusion assay.

2. Describe the orientation assay.

3. Describe in detail the pleiotropic phenotypes of farl-s alleles. Compare the phenotype of strains carrying the four different farl-s alleles described in Article 15 with wild-type FAR1 strains and with the phenotype of strains carrying other farl mutant alleles. Where possible, include each of the phenotypes listed below.

(a) Cell cycle arrest.

(b) Mating defects with different partners.

(c) FUS1 transcription.

(d) Farl protein expression.

(e) Mating confusion.

(f) Orientation to pheromone gradient.

4. Describe the experiments that demonstrate that Farlp has a function in mating in addition to its role in cell cycle arrest.

5. Describe the experiment and the analysis of the experimental results that allows the authors to conclude that the farl-s mutants orient towards the incipient bud site. What does this suggest with regard to the structure of the incipient bud site while the cell undergoes reorientation for schmoo formation?

6. Four farl-s alleles were isolated, sequenced, and characterized. Discuss the value of analyzing multiple alleles. How might their conclusions differ if mutant B4 were not available?

7. farl-60F3 complements farls-Dl. Assuming that the Farl protein does not form homomultimers, propose a mechanism for this intragenic complementation.

REFERENCES

Chang, F. (1991) Regulation of the cell cycle by a negative growth factor in yeast. Ph.D. Thesis, University of California, San Francisco.

Chenevert, J., N. Valtz, & I. Herskowitz (1994) Identification of genes required for normal pheromone-induced cell polarization in Saccharomyces cerevisiae. Genetics 136: 1287-1297.

Gartner, A., A. Jovanovic, D.I. Jeoung, S. Bourlat, F.R. Cross, & G. Ammerer (1998) Pheronome-dependent G1 cell cycle arrest requires Farl phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo. Mol. Cell. Biol. 18: 3681-3691.

Peter, M. & I. Herkowitz (1994) Direct inhibition of the yeast cyclin-dependent kinase Cdc28/ Cln2 by Farl. Science 265: 1228-1231.

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