Lac4 is the structural gene for beta-galactosidase in Kluyveromyces lactis

Abstract

Using genetic and biochemical techniques, we have determined that beta-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of beta-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac+ revertant, bearing lac4--14 and this unlinked suppressor, has subnormal levels of beta-galactosidase activity, and the Km for hydrolysis of o-nitrophenyl-beta, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of beta-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acrylamide gel electrophoresis shows that strains bearing lac4--23 and lac4--30 contain an inactive beta-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is observable in lac4--8, lac14--14 or lac4--29 mutants; (6) of all lac4 mutants, only those bearing lac4--23 or lac4--30 contain a protein that cross-reacts with anti beta-galactosidase antibody, a finding consistent with the previous result; and (7) beta-galactosidase activity in several Lac+ revertants of strains carrying lac4--23 or lac4--30 has greatly decreased thermostability.