Δ7-Sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles

Δ7-Sterol-C5-desaturase: molecular characterization and functional expression of wild-type and... An Arabidopsis thaliana recessive monogenic mutant (ste1-1) presenting a deficiency of the Δ7-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 Δ7-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 Δ7-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 Δ7-sterol-C5(6)-desaturases identified so far suggests that this amino acid is important for normal enzymatic function. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Δ7-Sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles

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Publisher
Springer Journals
Copyright
Copyright © 1999 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1006113919172
Publisher site
See Article on Publisher Site

Abstract

An Arabidopsis thaliana recessive monogenic mutant (ste1-1) presenting a deficiency of the Δ7-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 Δ7-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 Δ7-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 Δ7-sterol-C5(6)-desaturases identified so far suggests that this amino acid is important for normal enzymatic function.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 19, 2004

References

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