Delta7-sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles
| Autor: | T, Husselstein, H, Schaller, D, Gachotte, P, Benveniste |
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| Rok vydání: | 1999 |
| Předmět: |
Oxidoreductases Acting on CH-CH Group Donors
DNA Complementary Base Sequence DNA Plant Sequence Homology Amino Acid Genetic Complementation Test Molecular Sequence Data Arabidopsis Saccharomyces cerevisiae Gene Expression Regulation Enzymologic Blotting Southern Open Reading Frames Plants Toxic Gene Expression Regulation Plant Mutation Tobacco Humans Amino Acid Sequence Cloning Molecular Oxidoreductases Sequence Alignment Alleles |
| Zdroj: | Plant molecular biology. 39(5) |
| ISSN: | 0167-4412 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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