Isolation and Characterization of an Arabidopsis Thaliana cDNA Encoding a Delta 7-sterol-C-5-desaturase by Functional Complementation of a Defective Yeast Mutant

Plant J. 1996 Mar;9(3):391-8. doi: 10.1046/j.1365-313x.1996.09030391.x.

Abstract

A yeast null mutant (erg 3) defective in ERG 3, the gene encoding the C-5 sterol desaturase required for ergosterol synthesis was transformed with an Arabidopsis thaliana cDNA library inserted in a yeast vector. Transformants (4 x 10(5)) were screened for cycloheximide (CH) resistance and 400 possible clones were analyzed to determine their sterol profile. Low levels of ergosterol in addition to delta 7- and delta 8-sterols normally present in erg3 were isolated in three yeast transformants. Characterization of one transformant indicated a cDNA of 1141 bp. Transformation of an erg 3 strain with this plasmid led to CH resistance, nystatin sensitivity and an ergosterol profile. After subcloning in a pBluescript vector and subsequent sequencing, an ORF of 843 bp encoding a possible 281 amino acid polypeptide was deduced. Three histidine-rich motifs (HX3H, HX2HH and HX2HH) were found in the A. thaliana ORF which are also present in the yeast ERG 3 gene. These histidine-rich motifs are also characteristic of many membrane-bound fatty acid desaturases from higher plants. These data strongly suggest that the A. thaliana cDNA encodes a delta 7-sterol-C-5-desaturase.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / enzymology*
  • Arabidopsis / genetics*
  • Base Sequence
  • DNA, Complementary / genetics*
  • DNA, Complementary / isolation & purification
  • DNA, Plant / genetics*
  • DNA, Plant / isolation & purification
  • Genetic Complementation Test
  • Molecular Sequence Data
  • Mutation
  • Oxidoreductases / genetics*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Sequence Homology, Amino Acid
  • Transformation, Genetic

Substances

  • DNA, Complementary
  • DNA, Plant
  • Oxidoreductases
  • sterol delta-5 desaturase

Associated data

  • GENBANK/X90454