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Plant Molecular Biology

, Volume 30, Issue 6, pp 1139–1151 | Cite as

Molecular cloning, in vitro expression and characterization of a plant squalene synthetase cDNA

  • Kathleen M. Hanley
  • Olivier Nicolas
  • Timothy B. Donaldson
  • Constance Smith-Monroy
  • Gordon W. Robinson
  • Gary M. Hellmann
Research Article

Abstract

Squalene synthetase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the first committed step for sterol biosynthesis and is thought to play an important role in the regulation of isoprenoid biosynthesis in eukaryotes. Using degenerate oligonucleotides based on a conserved region found in yeast and human squalene synthetase genes, a cDNA was cloned from the plant Nicotiana benthamiana. The cloned cDNA contained an open reading frame of 1234 bp encoding a polypeptide of 411 amino acids (Mr 47002). Northern blot analysis of, poly(A)+ mRNA from N. benthamiana and N. tabacum cv. MD609 revealed a single band of ca. 1.6 kb in both Nicotiana species. The identity and functionality of the cloned plant squalene synthetase cDNA was further confirmed by expression of the cDNA in Escherichia coli and in a squalene synthetase-deficient erg9 mutant of Saccharomyces cerevisiae. Antibodies raised against a truncated form of the protein recognized an endogenous plant protein of appropriate size as well as the full-length bacterially expressed protein as detected by western analysis. Comparison of the deduced primary amino acid sequences of plant, yeast, rat and human squalene synthetase revealed regions of conservation that may indicate similar functions within each polypeptide.

Key words

cDNA Nicotiana PCR squalene synthetase 

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Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Kathleen M. Hanley
    • 1
  • Olivier Nicolas
    • 2
  • Timothy B. Donaldson
    • 2
  • Constance Smith-Monroy
    • 3
  • Gordon W. Robinson
    • 3
  • Gary M. Hellmann
    • 2
  1. 1.Biosource Technologies Inc.VacavilleUSA
  2. 2.R. J. Reynolds, Bowman Gray Technical CenterWinston-SalemUSA
  3. 3.Department of Metabolic DiseaseBristol-Myers Squibb Pharmaceutical Research InstitutePrincetonUSA

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