PHYSICAL AND FUNCTIONAL-CHARACTERIZATION OF THE CLONED LYS1-POMBE( GENE OF SCHIZOSACCHAROMYCES)

The alpha-aminoadipate pathway for the biosynthesis of lysine is prese nt in yeast and other higher fungi. The lys2 and lys5 mutants of Sacch aromyces cerevisiae as well as the lys1- and lys7- mutants of Schizosa charomyces pombe are blocked at the alpha-aminoadipate reductase step of this pathway. The cloned lys1+ gene in the plasmid pLYS1 isolated f rom a S.pombe genomic library complemented lys1- mutant of S. pombe. T he cloned L YS2 gene in the plasmid YEp620 and the LYS5 gene in the pl asmid pSC5 of S. cerevisiae exhibited heterologous complementation of lys1- and lys7- mutants. respectively, of S.pombe. The homologous lys1 + transformed cells exhibited five fold higher 2-aminoadipate reductas e activity while the heterologous lys1+ and lys7+ transformed cells ex hibited much less activity than the the wild type cells. The DNA inser t of the plasmid pLYS1 was determined to be 16.7 kb long and the lys1 gene has been subcloned within a 9.1 kb Clal-Clal DNA insert of the r ecombinant plasmids pLYS 1 B and pLYS 1 C. The restriction pattern for 12 enzymes of the 9.1 kb DNA insert, (Apal, Aval, BamHI, Clal, EcoRI, EcoRV, HindIII, Hpal, Pstl, Pvull. Sphl, and Xbal), exhibited no obvi ous similarity to that of the LYS2 gene of S. cerevisiae. A 1.7 kb Eco RI-HindIII DNA fragment of pLYS1B and pLYS1C complemented the lysl-131 mutation in an integrative transformation. Although the lys1+ gene of S. pombe is isofunctional to the L YS2 gene of S. cerevisiae, the res triction sites, and expression of these two genes exhibited considerab le divergence.