CLONING AND CHARACTERIZATION OF GNS1 - A SACCHAROMYCES-CEREVISIAE GENE INVOLVED IN SYNTHESIS OF 1,3-BETA-GLUCAN IN-VITRO

The GNS1 gene product is required for the synthesis of 1,3-beta-glucan in vitro, since mutations in this gene result in exhibit an 80 to 90% reduction in 1,3-beta-glucan synthase specific activity. gns1 mutant strains display a pleiotropic phenotype including resistance to a pneu mocandin B-0 analog (L-733,560), slow growth, and mating and sporulati on defects. The gns1-1 mutation was genetically mapped to within 1.35 centimorgans from the MAT locus on chromosome III. The wild-type GNS1 gene was isolated by complementing the pneumocandin resistance phenoty pe of the gns1-1 mutation and by hybridization with a chromosome III-d erived sequence being used as a probe. The nucleotide sequence of GNS1 was determined and compared with the homologous region of the chromos ome. The genetic and nucleotide sequence analyses revealed that GNS1 a nd the open reading frame, YCR34 [S. Oliver, Q. van der Aart, M. Agost oni-Carbone, and the Chromosome III Sequencing Group, Nature (London) 357:38-46, 1992], represent identical loci in the genome. Cells delete d for GNS1 are viable but exhibit slow growth as well as the pleiotrop ic phenotype of the gns1 mutants. The putative protein product is pred icted to be an integral membrane protein with five transmembrane helic es displaying an exoplasmic orientation for the N terminus and a cytop lasmic orientation for the C terminus. This protein may be a subunit o f 1,3-beta-glucan synthase.