Molecular cloning and characterization of the Candida albicans UB13 gene coding for a ubiquitin-hybrid protein

Using a polyubiquitin cDNA as a probe, we have isolated a clone (pPR3, a pE MBLYe23 derivative plasmid) containing the Candida albicans UB13 gene codin g for a fusion protein. This protein is formed by one ubiquitin subunit fus ed, at its C-terminus, to an unrelated peptide which is similar to the ribo somal protein encoded by the 3' tail of the Saccharomyces cerevisiae UB13 g ene. Southern blot analysis of chromosomal DNA probed with the 3' non-ubiqu itin tail of UBI3 indicated that only one homologous gene is present in the C, albicans genome. Heterelogous expression of pPR3 in a S. cerevisiae ubi 3 mutant strain complements the mutant phenotype (slow growth) conferred by the ubi3 defect; this provides direct evidence indicating that the clone c ontains 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; UBI 3 mRNA drops to undetectable levels on shifting the temperature of growing yeast cells from 28 degreesC to 42 degreesC. When Northern blot analysis wa s performed using a specific probe for the polyubiquitin (UBI4) gene, no dr op 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/o r enhanced mRNA decay), and suggest that UBI4 gene provides ubiquitin durin g the stress response. In addition, we failed to obtain C. albicans UBI3 nu ll mutant cells by sequential disruption of both alleles using the hisG::UR A3::hisG ('ura-blaster') cassette, suggesting that null mutants cells may b e unable to grow on selective media after transformation. The C, albicans U BI3 sequence has been deposited in the EMBL Data Library under Accession No . Y15608, Copyright (C) 2000 John Whey & Sons, Ltd.