A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning and characterization.
A phosphatidylinositol (PI) 3-kinase gene (CaVPS34) of the human pathogenic yeast Candida albicans was cloned by a PCR-based homology approach. The open reading frame encodes a 1020 amino acid protein with a calculated molecular weight of 118 kDa and a relative isoelectric point of 6.9. It shares 47% sequence identity with Saccharomyces cerevisiae Vps34p. Southern pattern indicated that CaVPS34 is probably present as a single copy gene per haploid genome in C. albicans. We localized the CaVPS34 gene on chromosome 1. Under all conditions tested a major CaVPS34 transcript of approximately 3. 5 kb could be detected. CaVPS34 mRNA levels increased during exponential growth up to 12-fold followed by a decline upon entry into stationary phase. The size of a 6xHis tag-CaVps34p fusion protein purified from Escherichia coli is in agreement with the calculated molecular mass of CaVps34p. It exhibits in vitro PI 3-kinase activity and produces only phosphatidylinositol 3-phosphate. The CaVPS34 gene under the control of its own promoter were not able to complement the temperature-sensitive growth of S. cerevisiae vps34. However, overexpression of CaVPS34 was sufficient to rescue the temperature-sensitive vps34 phenotype, suggesting a functional conservation in C. albicans.
Eck
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R
Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany. reck@pmail.hki-jena.de
Bruckmann
A
A
Wetzker
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R
Künkel
W
W
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002177:Candidiasis / Q000382:microbiology; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012331:RNA, Fungal / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D019869:Phosphatidylinositol 3-Kinases; D012331:RNA, Fungal