Physical and functional characterization of the cloned lys1+ gene of Schizosaccharomyces pombe.
The alpha-aminoadipate pathway for the biosynthesis of lysine is present in yeast and other higher fungi. The lys2 and lys5 mutants of Saccharomyces cerevisiae as well as the lys1- and lys7-mutants of Schizosacharomyces pombe are blocked at the alpha-aminoadipate reductase step of this pathway. The cloned lys1+ gene in the plasmid pLYS1 isolated from a S. pombe genomic library complemented lys1-mutant of S. pombe. The cloned LYS2 gene in the plasmid YEp620 and the LYS5 gene in the plasmid 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 alpha-aminoadipate reductase activity while the heterologous lys1+ and lys7+ transformed cells exhibited much less activity than the wild type cells. The DNA insert 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 recombinant plasmids pLYS1B and pLYS1C. 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 obvious similarity to that of the LYS2 gene of S. cerevisiae. A 1.7 kb EcoRI-HindIII DNA fragment of pLYS1B and pLYS1C complemented the lys1-131 mutation in an integrative transformation. Although the lys1+ gene of S. pombe is isofunctional to the LYS2 gene of S. cerevisiae, the restriction sites, and expression of these two genes exhibited considerable divergence.
Ford
R A
RA
Department of Microbiology, Miami University, Oxford, OH 45056.
Ye
Z H
ZH
Bhattacharjee
J K
JK
D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D003001:Cloning, Molecular; D005800:Genes, Fungal / Q000235:genetics*; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000235:genetics*
D000445:Aldehyde Oxidoreductases; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase