Cloning and characterisation of the HYR1 and UB14 genes from Candida albicans
The ability to grow as either an ellipsoidal budding yeast or an elongate hypha is a potentially important virulence determinant of the fungal pathogen Candida albicans. This morphological transition also provides an interesting example of cellular differentiation in a simple eukaryote. We have elected to study this morphological change by isolating and analysing genes which are regulated during the yeast-to-hypha transition. An unusual chimeric cDNA isolated from a C. albicans cDNA expression library was shown to contain an in vitro fusion of two unlinked sequences. The 5' end of the clone exhibited 86% nucleotide sequence identity to the Saccharomyces cerevisiae polyubiquitin gene and was therefore designated caUB14. The 3' end of the clone contained a previously unidentified ORF which was demonstrated, by northern analysis, to be expressed exclusively during hypha formation in C. albicans. This gene was therefore designated HYR1, reflecting its Hyphal regulation. Genomic clones representing the full length genes for HYR1, and caUB14 were isolated and the full sequence of each generated. Southern and CHEF analysis were used to demonstrate that each gene is present at a single locus in the C. albicans genome, located on chromosomes 3 and 1, respectively. A combination of PCR and northern analysis was used to show that there is considerable heterogeneity in the lengths of caUB14 genes between different C. albicans strains and other Candida species. To establish a role for the Hyr1p protein in C. albicans hyr1/hyr1 null mutant and a mutant over-expressing the HYR1 gene were created. Comparative analysis of growth rate, hypha formation, adherence and the susceptibility to cytotoxic compounds between the mutants and parental strain failed to reveal any significant phenotype that might suggest a cellular function for Hyr1p. Microscopic analysis of yeast and hyphal cells and of colony morphology also showed no differences between the strains. However, preliminary data on virulence in a systemic mouse model suggest that the null mutant may be more virulent than the wild type strain.