Cloning and biochemical characterization of LYS5 gene of Saccharomyces cerevisiae

• Published in: Current genetics Vol. 13; no. 4; pp. 299 - 304
• Main Authors: Borell, C.W, Bhattacharjee, J.K
• Format: Journal Article
• Language: English
• Published: United States 01.04.1988
• Subjects: Aldehyde Oxidoreductases - genetics; alpha-aminoadipate reductase; characterization; cloning; Cloning, Molecular; DNA Restriction Enzymes; enzyme activity; Genes; Genes, Fungal; Genotype; L-Aminoadipate-Semialdehyde Dehydrogenase; Plasmids; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics
• ISSN: 0172-8083; 1432-0983
• DOI: 10.1007/BF00424423

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.