A SYSTEM FOR GENE CLONING AND MANIPULATION IN THE YEAST CANDIDA-GLABRATA

The opportunistic pathogenic yeast, Candida (Torulopsis) glabrata, is an asexual imperfect fungus that exists largely as a haploid. Besides being a clinically important pathogen, this yeast also provides a mode l system for understanding basic biological mechanisms such as metal-a ctivated metallothionein-encoding gene transcription. To facilitate mo lecular genetic studies in C. glabrata, we isolated a strain auxotroph ic for uracil biosynthesis. The ura(-) mutation could be functionally complemented by the URA3 gene of Saccharomyces cerevisiae, consistent with a defect in the C. glabrata URA3 gene in this strain. We also fou nd that the centromere-based S. cerevisiae plasmid pRS316 could stably transform and replicate in multiple copies in C. glabrata. In contras t, high-copy-number S. cerevisiae plasmids containing the 2 mu circle autonomous replication sequence were not able to replicate productivel y in C. glabrata. We cloned the C. glabrata URA3 gene, encoding orotid ine-5'-phosphate decarboxylase, by complementation of a ura3(-) strain of S. cerevisiae. The deduced amino-acid sequence is highly similar t o that of the URA3 protein from S. cerevisiae. C. glabrata URA3 provid es a genetic locus for targeted gene integration in C. glabrata. Integ rative plasmids were constructed based on the cloned C. glabrata URA3 and are applicable for directed insertions of genes of interest at the ura3 locus through homologous recombination.