Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.
A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.
Lorenz
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Department of Microbiology, North Carolina State University, Raleigh 27695-7615.
Parks
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004352:Drug Resistance, Microbial / Q000235:genetics; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000378:metabolism; D015723:Gene Library; D016153:Genes, Suppressor; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D004271:DNA, Fungal; D010088:Oxidoreductases; C022672:delta(14)-sterol reductase; C015291:ignosterol; D004875:Ergosterol