A system for gene cloning and manipulation in the yeast Candida glabrata.

Published

Journal Article

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 model system for understanding basic biological mechanisms such as metal-activated metallothionein-encoding gene transcription. To facilitate molecular genetic studies in C. glabrata, we isolated a strain auxotrophic 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 found that the centromere-based S. cerevisiae plasmid pRS316 could stably transform and replicate in multiple copies in C. glabrata. In contrast, high-copy-number S. cerevisiae plasmids containing the 2 mu circle autonomous replication sequence were not able to replicate productively in C. glabrata. We cloned the C. glabrata URA3 gene, encoding orotidine-5'-phosphate decarboxylase, by complementation of a ura3- strain of S. cerevisiae. The deduced amino-acid sequence is highly similar to that of the URA3 protein from S. cerevisiae. C. glabrata URA3 provides a genetic locus for targeted gene integration in C. glabrata. Integrative 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.

Full Text

Duke Authors

Cited Authors

  • Zhou, P; Szczypka, MS; Young, R; Thiele, DJ

Published Date

  • May 3, 1994

Published In

Volume / Issue

  • 142 / 1

Start / End Page

  • 135 - 140

PubMed ID

  • 8181748

Pubmed Central ID

  • 8181748

International Standard Serial Number (ISSN)

  • 0378-1119

Digital Object Identifier (DOI)

  • 10.1016/0378-1119(94)90368-9

Language

  • eng

Conference Location

  • Netherlands