Cloning and biochemical characterization of LYS5 gene of Saccharomyces cerevisiae

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Abstract
In Saccharomyces cerevisiae, the functions of two unlinked genes (LYS2 and LYS5) are required for the synthesis of the lysine biosynthetic enzyme, α-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 α-aminoadipate reductase activity, whereas lys5 mutant and plasmid-cured transformed strain exhibited none. Lys2 + transformants consistently had five times greater α-aminoadipate reductase activity when compared with the wildtype and the Lys5 + transformant. The α-aminoadipate reductase activity was repressed in lysine-grown wildtype and Lys5 + transformed cells but not in Lys2 + transformed cells. A Lys2 + and Lys5 + double transformant exhibited higher a-aminoadipate reductase activity than lys2 + or lys5 + transformant.