Cloning and biochemical characterization of LYS5 gene of Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the functions of two unlinked genes (LYS2 and LYS5) are required for the synthesis of the lysine biosynthetic enzyme, alpha-aminoadipate reductase. The LYS5 gene of S. cerevisiae was cloned by functional complementation of a lys5 mutant, X4004-3A, using a YEp24 plasmid library. The cloned LYS5 gene was contained within a 7.5 kb DNA insert of the recombinant plasmid pSC5. Cloning of LYS5 gene was confirmed by second cycle transformation of a lys5 mutant with the pSC5 plasmid, growth response studies, and plasmid loss experiments with Lys5+ transformants. Analysis of restriction digests of the pSC5 plasmid revealed 3 EcoRI, 5 PvuII, 1 PstI, 1 BglII and 2 HpaI sites in the 7.5 kb insert. A 3.9 kb internal pSC5 fragment hybridized only to the plasmid pSC5, but no homology was observed with LYS2 DNA or the YEp24 vector. The pSC5 transformed Lys5+ cells and the wild-type strain exhibited same level of alpha-aminoadipate reductase activity, whereas lys5 mutant and plasmid-cured transformed strain exhibited none. Lys2+ transformants consistently had five times greater alpha-aminoadipate reductase activity when compared with the wild-type and the Lys5+ transformant. The alpha-aminoadipate reductase activity was repressed in lysine-grown wild-type and Lys5+ transformed cells but not in Lys2+ transformed cells. A Lys2+ and Lys5+ double transformant exhibited higher alpha-aminoadipate reductase activity than lys2+ or lys5+ transformant.