Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae

DNA Cell Biol. 1992 Nov;11(9):685-92. doi: 10.1089/dna.1992.11.685.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA, Fungal
  • Drug Resistance, Microbial / genetics
  • Ergosterol / analogs & derivatives*
  • Ergosterol / metabolism
  • Gene Library
  • Genes, Suppressor
  • Genome, Fungal
  • Molecular Sequence Data
  • Mutagenesis
  • Open Reading Frames
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Restriction Mapping
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*

Substances

  • DNA, Fungal
  • Oxidoreductases
  • delta(14)-sterol reductase
  • ignosterol
  • Ergosterol

Associated data

  • GENBANK/S44913
  • GENBANK/S44916
  • GENBANK/S44919
  • GENBANK/S44921
  • GENBANK/S44927
  • GENBANK/S44929
  • GENBANK/S44930
  • GENBANK/S44931
  • GENBANK/S44934
  • GENBANK/S49653