Isolation and characterization of an Arabidopsis thaliana cDNA encoding a delta 7-sterol-C-5-desaturase by functional complementation of a defective yeast mutant

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.