Elsevier

Gene

Volume 137, Issue 2, 31 December 1993, Pages 179-185
Gene

Cloning of the dihydroxyacid dehydratase-encoding gene (ILV3) from Saccharomyces cerevisiae

https://doi.org/10.1016/0378-1119(93)90004-MGet rights and content

Abstract

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or α-ketobutyrate are converted into α-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes (ILV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (cM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ILV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILV genes have been cloned, thus allowing direct molecular analyses of their regulation.

References (35)

  • R. Dumas et al.

    Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts

    Biochem. J.

    (1991)
  • R.T. Elder et al.

    RNA from yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA

  • S.C. Falco et al.

    Genetic analysis of mutants of Saccharomyces cerevisiae resistant to the herbicide sulfometuron methyl

    Genetics

    (1985)
  • S.C. Falco et al.

    Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase

    Nucleic Acids Res.

    (1985)
  • J.W. Fickett

    Recognition of protein coding regions in DNA sequences

    Nucleic Acids Res.

    (1982)
  • J.J. Godon et al.

    Branched-chain amino acid biosynthesis genes in Lactococcus lactis subsp. lactis

    J. Bacteriol.

    (1992)
  • J.A. Grandoni et al.

    Transcriptional regulation of the ilv-leu operon of Bacillus subtilis

    J. Bacteriol.

    (1992)
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    ∗∗

    Division of Immunology, La Jolla Institute for Allergy and Immunology, 11149 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel. (1-619) 558-3500.

    Present addresses: Center for Biologies Evaluation and Research, F.D.A N.I.H., Bldg. 29, 8800 Rockville Pike, Bethesda, MD 20892, USA. Tel. (1-301) 496-4038.

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