MOLECULAR-CLONING, IN-VITRO EXPRESSION AND CHARACTERIZATION OF A PLANT SQUALENE SYNTHETASE CDNA

Squalene synthetase (farnesyl-diphosphate:farnesyl-diphosphate farnesy ltransferase, EC 2.5.1.21) catalyzes the first committed step for ster ol biosynthesis and is thought to play an important role in the regula tion of isoprenoid biosynthesis in eukaryotes. Using degenerate oligon ucleotides based on a conserved region found in yeast and human squale ne synthetase genes, a cDNA was cloned from the plant Nicotiana bentha miana. The cloned cDNA contained an open reading frame of 1234 bp enco ding a polypeptide of 411 amino acids (M(r) 47002). Northern blot anal ysis of poly(A)(+) mRNA from N. benthamiana and N. tabacum cv. MD609 r evealed a single band of ca. 1.6 kb in both Nicotiana species. The ide ntity and functionality of the cloned plant squalene synthetase cDNA w as further confirmed by expression of the cDNA in Escherichia coli and in a squalene synthetase-deficient erg9 mutant of Saccharomyces cerev isiae. Antibodies raised against a truncated form of the protein recog nized an endogenous plant protein of appropriate size as well as the f ull-length bacterially expressed protein as detected by western analys is. Comparison of the deduced primary amino acid sequences of plant, y east, rat and human squalene synthetase revealed regions of conservati on that may indicate similar functions within each polypeptide.