Isolation and characterization of an Arabidopsis thaliana cDNA encoding a delta 7-sterol-C-5-desaturase by functional complementation of a defective yeast mutant.
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.
Gachotte
D
D
Département de Biologie Cellulaire et Moléculaire, Institut de Botanique, Strasbourg, France.
Husselstein
T
T
Bard
M
M
Lacroute
F
F
Benveniste
P
P
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics* / Q000302:isolation & purification; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D018076:DNA, Complementary; D018744:DNA, Plant; D010088:Oxidoreductases; C039306:sterol delta-5 desaturase