Molecular genetics of a Candida albicans-specific DNA fragment.

摘要

Candida albicans is an opportunistic fungal pathogen affecting increasing numbers of immunocompromised, at-risk patients. Efforts toward understanding C. albicans are focused on significant clinical questions concerning diagnosis, pathogenesis, and epidemiology of candidiasis, and also, developmental components and control of C. albicans morphogenesis. Molecular genetic studies hold promise for providing insight into this complex polymorphic organism.; The fungal cell wall acts as a barrier to extraction of high quality DNA for molecular analysis. In C. albicans studies reported here, Ribi Cell fractionation, autolysis, and spheroplast DNA extraction methods were equivalent at producing high molecular weight fragments that were susceptible to enzymatic manipulation. The distribution of MspI-digested (C{dollar}downarrow{dollar}CGG) C. albicans DNA contained unexpectedly large molecular weight fragments. Some MspI fragments larger than 4 kbp appeared repetitive by fluorescent staining intensity, but were not ribosomal or mitochondrial in origin.; Attempts to clone repetitive fragments led to the identification of a C. albicans and C. stellatoidea species-specific MspI/HpaII DNA fragment (Cutler, J. E., P. M. Glee, and H. L. Horn. 1988. J. Clin, Microbiol. 26:1720-1724). The 3.87 kbp C. albicans fragment did not hybridize to DNA from other Candida species including C. krusei, C. tropicalis, C. kefyr, C. parapsilosis, C. guilliermondii, or C. lusitaniae. Southern blot hybridization was extended to S. cerevisiae, N. crassa, Torulopsis, Gaeumanomyces gramminis, Aspergillus, Mucor, Histoplasma, Sepedonium, Chrysosporium, Coccidioides, Cryptococcus, Blastomyces, Trichomonas vaginalis, and to human and mouse DNA. The C. albicans specific fragment did not hybridize to any of these DNAs and may be useful as a DNA diagnostic probe. The C. albicans species-specific fragment indicated the following: 1-2 copies per haploid genome in DNA dot blots; 4 different MspI band patterns for 50 C. albicans isolates; no methylation changes at fragment-terminal CCGG sites during yeast to mycelial transition; partial sequence data revealed A-T rich regions; and positive hybridization to Northern blots of yeast and mycelial form RNA indicated transcribed sequences.; Two other MspI/HpaII C. albicans DNA pieces were partially characterized by sequencing and both indicated positive hybridization to Northern blots of C. albicans RNA. One fragment showed potential for strain differentiation and multi-chromosomal hybridization.