Isolation and characterization of an Arabidopsis thaliana cDNA encoding a Δ7-sterol-C-5-desaturase by functional complementation of a defective yeast mutant

✍ Scribed by Daniel Gachotte; Tania Husselstein; Martin Bard; François Lacroute; Pierre Benveniste

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 518 KB
• Volume: 9
• Category: Article
• ISSN: 0960-7412
• DOI: 10.1046/j.1365-313x.1996.09030391.x

✦ Synopsis

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 (4×10^5^) were screened for cycloheximide (CH) resistance and 400 possible clones were analyzed to determine their sterol profile. Low levels of ergosterol in addition to Δ^7^‐ and Δ^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 (HX~3~H, HX~2~HH and HX~2~HH) 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 Δ^7^‐sterol‐C‐5‐desaturase.