Molecular cloning of the secretory acid proteinase gene from Candida albicans and its use as a species-specific probe.
Candida albicans secretes an acid proteinase when grown with a protein as a sole nitrogen source. The gene encoding this proteinase was isolated from a genomic expression library of C. albicans constructed in lambda gt11 by screening with antiproteinase antibodies. The affinity-purified antibodies used to verify the clones are monspecific; these do not cross-react with any other protein in the culture supernatants or crude extracts of C. albicans but strongly react with fusion proteins encoded by recombinant clones, revealing that these are true proteinase clones. Genomic Southern blot analysis shows that the proteinase gene is present at a unique locus and that there is no other closely related gene in the C. albicans genome. The proteinase gene probe identified two transcripts on Northern blots (RNA blots), which are present at a much higher level in C. albicans cells induced for proteinase secretion than in uninduced cells. The aspartyl proteinase gene reported earlier (T.J. Lott, L.S. Page, P. Boiron, J. Benson, and E. Reiss, Nucleic Acids Res. 17:1779, 1989) is not that of secretory acid proteinase, since the N-terminal amino acid sequence of secretory acid proteinase does not correspond to the deduced amino acid sequence of the aspartyl proteinase gene. The secretory acid proteinase gene was used to probe Southern blots of genomic DNA of several medically important Candida species and Saccharomyces cerevisiae. Under hybridization and wash conditions of low stringency, Candida tropicalis and Candida parapsilosis, in addition to C. albicans strains, gave specific signals, implying that C. tropicalis and C. parapsilosis have homologous secretory acid proteinase genes. However, under under wash conditions of high stringency, signals were obtained only with C. albicans strains, suggesting that this gene can be used as a species-specific probe. A simple yeast colony hybridization technique is sufficient to distinguish C. albicans from other yeasts.
Ganesan
K
K
Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Banerjee
A
A
Datta
A
A
D000818:Animals; D000908:Antibodies, Fungal / Q000276:immunology; D015152:Blotting, Northern; D015139:Blotting, Southern; D002176:Candida albicans / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D015342:DNA Probes; D004271:DNA, Fungal / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D009693:Nucleic Acid Hybridization; D011817:Rabbits; D012697:Serine Endopeptidases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000908:Antibodies, Fungal; D015342:DNA Probes; D004271:DNA, Fungal; D012697:Serine Endopeptidases