Delta7-sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles.
An Arabidopsis thaliana recessive monogenic mutant (ste1-1) presenting a deficiency of the delta7-sterol-C5(6)-desaturase step in the sterol pathway has been reported previously [12]. To further characterize ste1-1, Arabidopsis, Nicotiana tabacum and Homo sapiens cDNAs encoding delta7-sterol-C5(6)-desaturases were isolated and identified on the basis of their ability to restore ergosterol synthesis in erg3, a yeast null mutant whose gene encoding the delta7-sterol-C5(6)-desaturase was disrupted. Overexpression of the Arabidopsis cDNA driven by a 35S promoter in transgenic ste1-1 plants led to full complementation of the mutant. This result demonstrates that STE1 was the impaired component in the desaturation system. Four independent reverse transcriptions of ste1-1 RNA followed by polymerase chain reactions (RT-PCRs), yielded a single product. Alignment of the wild-type ORF with the RT-PCR derived ste1-1 ORF revealed a single amino acid substitution: Thr-114 in the wild-type is changed to Ile in ste1-1. Expression in erg3 resulted in a 6-fold lowered efficiency of the ste1-1 ORF in complementing the yeast biosynthetic pathway when compared to the wild-type ORF. The presence of this mutation in the mutant ste1-1 genomic sequence (and no additional modification between ste1-1 and wild-type genes) demonstrates that the change of the Thr-114 to Ile is necessary and sufficient to create the leaky allele ste1-1. The occurrence of a hydroxylated amino acid (Thr or Ser) at the position corresponding to Thr-114 in the five delta7-sterol-C5(6)-desaturases identified so far suggests that this amino acid is important for normal enzymatic function.
Husselstein
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Institut de Biologie Moléculaire des Plantes, Département d'Enzymologie Cellulaire et Moleculaire, UPR 406 du CNRS, Strasbourg, France.
Schaller
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Gachotte
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Benveniste
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D000483:Alleles; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010947:Plants, Toxic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000737:chemistry / Q000201:enzymology / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; C033144:lathosterol delta-5-dehydrogenase; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C039306:sterol delta-5 desaturase