Topics of current studies include: 1) Cloning of the URA5 gene and development of a transformation system in Cryptococcus neoformans; 2) Characterization of stable and unstable transformants of C neoformans; 3) Cloning and sequencing of a regulatory gene for alpha glucosidase in Candida albicans; 4) Linkage mapping of C albicans chromosomes. A cDNA encoding orotidine monophosphate pyrophosphorylase (OMPP, URA5) of Cryptococcus neoformans var. neoformans has been isolated by complementation of E. coli pyrE mutants. The OMPP cDNA was then used as a probe to isolate a genomic DNA fragment containing the entire URA5 gene. The plasmid, pURA5g2, containing the URA5 gene was introduced into a C neoformans ura5 strain by electroporation. The transformants were of three kinds: stable transformants showing homologous or ectopic integration of pURA5g2, stable transformants showing integration as well as having autonomously replicating plasmids, and unstable transformants containing only autonomously replicating plasmids. A Candida albicans gene involved in sucrose utilization was cloned and the gene was found to complement the sucrose utilization defect of Type II C. stellatoidea. The DNA sequence showed that it encodes an open reading frame of 501 amino acids. No significant homology was found to any protein in the PRI data base. A single zinc finger motif was found at the amino terminus, suggesting that this protein has a regulatory function. Separation of C. albicans chromosomes using contour clamped homogeneous electric field (CHEF) electrophoresis showed 8 bands instead of 7 as previously believed. Numerous cloned genes were used as probes to reveal the linkage group. Several genes previously thought to be located on chromosome 1 were found to hybridize to chromosome 2.
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