Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae.

Abstract

During sporulation of Saccharomyces cerevisiae, the four haploid nuclei generated by meiosis are encapsulated within multilayered spore walls. Taking advantage of the natural fluorescence imparted to yeast spores by the presence of a dityrosine-containing macromolecule in the spore wall, we identified and cloned two genes, termed DIT1 and DIT2, which are required for spore wall maturation. Mutation of these genes has no effect on the efficiency of spore formation or spore viability. The mutant spores, however, fail to accumulate the spore wall-specific dityrosine and lack the outermost layer of the spore wall. The absence of this cross-linked surface layer reduces the resistance of the spores to lyric enzymes, to ether, and to elevated temperature. Expression of the DIT and DIT2 genes is restricted to sporulating cells, with the DIT1 transcripts accumulating at the time of prospore enclosure and just prior to the time of dityrosine biosynthesis. Both genes act in a sporeautonomous manner implying that at least some of the activities responsible for forming the outermost layer of the spore wall reside within the developing spore rather than in the surrounding ascal cytoplasm. As the DIT2 gene product has significant homology with cytochrome P-450s, DIT2 may be responsible for catalyzing the oxidation of tyrosine residues in the formation of dityrosine.

Mutation of the SPS1-encoded protein kinase of Saccharomyces cerevisiae leads to defects in transcription and morphology during spore formation.

“…3A); expression of HOPl preceded expression of SPSl by ~2 hr, and expres sion of DITl began 2 hr after the SPSl gene had been turned on. This sequential pattern of gene expression was consistent with previous observations (Briza et al 1990b;Mitchell et al 1990;Smith et al 1990). …”

“…HOPl, which is is required for pairing of homologous chromosomes (Hollingsworth and Byers 1989;Hollingsworth et al 1990), is expressed coincidentally with a large number of other meiotic genes such as spoil (Atcheson et al 1987), REDl (Thompson and Roeder 1989), and REC114 (Pittman et al 1993), which encode products required for successful comple tion of meiosis (for review, see Mitchell 1994). DITl, which is expressed just prior to spore formation, is re quired for deposition of the outermost layer of the spore wall (Briza et al 1990b). We found that HOPl, SPSl, and DITl were turned on sequentially (Fig.…”

“…6C), which appears as a very thin, osmiophilic layer, consists of a crosslinked insoluble macromolecule containing a large amount of dityrosine (Briza et al 1990a,b). This layer, which is responsible for the resistance of spores to degradative enzymes and organic solvents (Briza et al 1990b) is closely associated, perhaps by covalent link ages, with an underlying chitosan layer (Briza et al 1988), which appears as a more diffuse osmiophilic layer. The innermost layers of the spore wall are similar in composition to the vegetative cell wall and often appear as a single electron-transparent layer (Katohda et al 1984;Briza et al 1988).…”

“…Various genetic screens have led to the isolation of mu tants that are defective in meiotic recombination, homolog pairing, segregation of chromosomes, and spore formation (for examples, see Esposito 1969, 1974;Tsuboi 1983;Rockmill and Roeder 1988;HoUingsworth and Byers 1989;Briza et al 1990b;Malone et al 1991). Cloning and characterization of a large number of the genes identified in these studies have almost invari ably revealed that they are expressed preferentially in sporulating cells (for review, see Mitchell 1994).…”

Yeast ascospore wall assembly requires two chitin deacetylase isozymes

“…The two outer layers confer on the spores the characteristic resistance against environmental stress. [1,2] Chitin deacetylase (CDA) is the enzyme that catalyzes the conversion of chitin to chitosan by deacetylation of N-acetyl-D-glucosamine residues. So far, CDA enzymes have been isolated from the fungi Mucor rouxii and Colletotrichum lindemuthianum [3,4] and genes encoding CDA have been studied in M. rouxii and Saccharomyces cerevisiae [5,6].…”