Molecular cloning and characterization of the Candida albicans UBI3 gene coding for a ubiquitin-hybrid protein

Using a polyubiquitin cDNA as a probe, we have isolated a clone (pPR3, a pEMBLYe23 derivative plasmid) containing the Candida albicans UBI3 gene coding for a fusion protein. This protein is formed by one ubiquitin subunit fused, at its C-terminus, to an unrelated peptide which is similar to the ribosomal protein encoded by the 3' tail of the Saccharomyces cerevisiae UBI3 gene. Southern blot analysis of chromosomal DNA probed with the 3' non-ubiquitin tail of UBI3 indicated that only one homologous gene is present in the C. albicans genome. Heterelogous expression of pPR3 in a S. cerevisiae ubi3 mutant strain complements the mutant phenotype (slow growth) conferred by the ubi3 defect; this provides direct evidence indicating that the clone contains the C. albicans UBI3 gene Northern blot analysis showed that UBI3 gene is expressed in yeast and germ-tube cells of C. albicans, although the UBI3 mRNA levels in starved yeast cells are below the detection limit; UBI3 mRNA drops to undetectable levels on shifting the temperature of growing yeast cells from 28 degrees C to 42 degrees C. When Northern blot analysis was performed using a specific probe for the polyubiquitin (UBI4) gene, no drop in the mRNA levels was detected following thermal upshift or in starved cells. These results indicate that stress conditions (starvation or thermal upshift) negatively regulate UBI3 expression (transcriptional arrest and/or enhanced mRNA decay), and suggest that UBI4 gene provides ubiquitin during the stress response. In addition, we failed to obtain C. albicans UBI3 null mutant cells by sequential disruption of both alleles using the hisG::URA3::hisG ('ura-blaster') cassette, suggesting that null mutants cells may be unable to grow on selective media after transformation.