知花 博治

<title>ABSTRACT</title> The major repeat sequence (MRS) is found at least once on all but one chromosome in <italic>Candida albicans</italic>, but as yet it has no known relation to the phenotype. The MRS affects karyotypic variation by serving as a hot spot for chromosome translocation and by expanding and contracting internal repeats, thereby changing chromosome length. Thus, MRSs on different chromosomes and those on chromosome homologues can differ in size. We proposed that the MRS's unique repeat structure and, more specifically, the size of the MRS could also affect karyotypic variation by altering the frequency of mitotic nondisjunction. Subsequent analysis shows that both natural and artificially induced differences in the size of the chromosome 5 MRS can affect chromosome segregation. Strains with chromosome 5 homologues that differ in the size of the naturally occurring MRSs show a preferential loss of the homologue with the larger MRS on sorbose, indicating that a larger MRS leads to a higher risk of mitotic nondisjunction for that homologue. While deletion of an MRS has no deleterious effect on the deletion chromosome under normal growth conditions and leads to no obvious phenotype, strains that have the MRS deleted from one chromosome 5 homologue preferentially lose the homologue with the MRS remaining. This effect on chromosome segregation is the first demonstration of a phenotype associated with the MRS.

<title>ABSTRACT</title> Parasexual genetic analysis of <italic>Candida albicans</italic>utilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced the<italic>IMH3</italic> ^r gene from <italic>C. albicans</italic> strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213–218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331–2338, 1997) have shown that the wild-type<italic>IMH3</italic> gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that the<italic>IMH3</italic> ^r gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of <italic>C. albicans</italic> and <italic>Candida tropicalis</italic>. We also identified the presence of autonously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a φ-type long terminal repeat element outside the <italic>IMH3</italic> open reading frame of the gene in some strains. We used the <italic>IMH3</italic> ^r allele to disrupt one allele of <italic>ARG4</italic> in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.

We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation. The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae. The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S. cerevisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system. We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening. Copyright (C) 2000 Elsevier Science B.V.

Pulse-chase experiments indicated that the extracellular proteinase (EPR) of Candida albicans originates as a 45 kDa precursor protein which is processed to a 43 kDa protein prior to secretion. Secretion was routinely stimulated in EPR induction medium which contains bovine serum albumin (BSA) and glucose. Although EPR was not induced without glucose as a carbon source, EPR secretion was induced without the addition of BSA or other nitrogen sources. Furthermore, it was shown that EPR production was not induced at pH > 6.0, irrespective of the presence of a nitrogen source. This suggests that medium pH may act directly upon EPR induction, and not as a secondary effect of the nitrogen supply from EPR-mediated protein digestion, which exhibited a pH optimum of around pH 3.5. When germ tube induced cells were transferred to EPR induction medium, EPR was not induced. Thus, EPR production and germ tube formation may not be induced by the same conditions. We speculate that EPR production and germ tube formation do not co-operate in the invasive process but play different and separate roles.