Abstract. The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chsl) and two of them deficient in either Chs3 (calRI mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the

Cyclins regulate the major cell cycle transitions in eukaryotes through association with cyclin-dependent protein kinases (CDKs). In yeast, G1 cyclins are essential, rate-limiting activators of cell cycle initiation. G1-specific accumulation of one G1 cyclin, Cln2, results from periodic gene expression coupled with rapid protein turnover. Site-directed mutagenesis of CLN2 revealed that its phosphorylation provides a signal that promotes rapid degradation. Cln2 phosphorylation is dependent on the Cdc28 protein kinase, the CDK that it activates. These findings suggest that Cln2 is rendered self-limiting by virtue of its ability to activate its cognate CDK subunit.

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter ceUls caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescehce microscopy, in a S. cerevisiae wild-type cells during a-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglitinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in a-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five-to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and 1-glucan in wild-type and m,utant strains were similar; however, the percentage of ,B-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to,a cell wall enzymic lytic complex were rather similar.The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.

Abstract. The CALI gene was cloned by complementation of the defect in Calcofluor-resistant calR1 mutants of Saccharomyces cerevisiae. Transformation of the mutants with a plasmid carrying the appropriate insert restored Calcofluor sensitivity, wild-type chitin levels and normal spore maturation. Southern blots using the DNA fragment as a probe showed hybridization to a single locus. Allelic tests indicated that the cloned gene corresponded to the calRI locus . The DNA insert contains a single open-reading frame encoding a protein of 1,099 amino acids with a molecular mass of 124 kD. The predicted amino acid sequence shows several regions of homology with those of chitin synthases 1 and 2 from S. cerevisiae and chitin syn-HE antifungal effect of Calcofluor White (33, 34), a substance that binds specifically to chitin in vivo (10), was used to isolate and characterize several Saccharo myces cerevisiae mutants resistant to this fluorochrome (35) . The most interesting feature of these mutants was a defect in chitin synthesis in vivo not only during the vegetative cycle but also in response to the pheromone a-factor (35) . A defect in spore maturation in the homozygous condition was observed (35). The possibility that these mutants could define physiologically relevant genes involved in chitin synthesis was apparent; therefore, the cloning and sequencing of the gene corresponding to one of them, cal"l, was undertaken . In this report we show that the protein sequence encoded by the cloned gene (CAI/) has significant similarities with those of chitin synthase 1 (9) and chitin synthase 2 (42, 43) . Recently, Bulawa and Osmond (8) detected the presence of a third chitin synthase (chitin synthase 3 or Chs31) in strains defective in chitin synthase 1 and 2. We report here that Reprint requests should be addressed to Enrico Cabib, National Institutes of Health, Building 10, Room 9N-115, Bethesda, MD 20892 .A preliminary report of parts of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology held at The Hague, Netherlands, 1990.1. Abbreviations used in this paper: Chsl-3, chitin synthase 1-3 ; canChsl, chitin synthase 1 from Candida albicans .© The Rockefeller University Press, 0021-9525/91/07/101/9 $2 .00 The Journal of Cell Biology, Volume 114, Number 1, July 1991101-109 thase 1 from Candida albicans . calR1 mutants have been found to be defective in chitin synthase 3, a trypsin-independent synthase . Transformation of the mutants with a plasmid carrying CALI restored chitin synthase 3 activity ; however, overexpression of the enzyme was not achieved even with a high copy number plasmid . Since Calcofluor-resistance mutations different from calRI also result in reduced levels of chitin synthase 3, it is postulated that the products of some of these CAL genes may be limiting for expression of the enzymatic activity. Disruption of the CALI gene was not lethal, indicating that chitin synthase 3 is not an essential enzyme for S. cerevisiae. ca1R1 mutants are deficient in chiti...