Cloning and sequencing of the Candida albicans C-4 sterol methyl oxidase gene (ERG25) and expression of an ERG25 conditional lethal mutation in Saccharomyces cerevisiae.
The ERG25 gene encoding the Candida albicans C-4 sterol methyl oxidase was cloned and sequenced by complementing a Saccharomyces cerevisiae erg25 mutant with a C. albicans genomic library. The Erg25p is comprised of 308 amino acids and shows 65 and 38% homology to the enzymes from S. cerevisiae and Homo sapiens, respectively. The protein contains three histidine clusters common to nonheme iron-binding enzymes and an endoplasmic reticulum retrieval signal as do the proteins from S. cerevisiae and humans. A temperature-sensitive (ts) conditional lethal mutation of the C. albicans ERG25 was isolated and expressed in S. cerevisiae. Sequence analysis of the ts mutant indicated an amino acid substitution within the region of the protein encompassed by the histidine clusters involved in iron binding. Results indicate that plasmid-borne conditional lethal mutants of target genes have potential use in the rescue of Candida mutations in genes that are essential for viability.
Kennedy
M A
MA
Department of Biology, Indiana University Purdue University Indianapolis, 46202-5132, USA.
Johnson
T A
TA
Lees
N D
ND
Barbuch
R
R
Eckstein
J A
JA
Bard
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular; D004926:Escherichia coli; D005804:Genes, Lethal; D015698:Genomic Library; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013261:Sterols / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D013261:Sterols; D006899:Mixed Function Oxygenases; C027532:methylsterol monooxygenase