LAC4 IS THE STRUCTURAL GENE FOR BETA-GALACTOSIDASE IN KLUYVEROMYCES-LACTIS

Abstract

B-Galactosidase in the yeast K. lactis is coded by the LAC4 locus. 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. Three of 5 lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA. 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. The level of .beta.-galactosidase activity per cell is directly proportional to the number of copies of LAC4. Analysis of cell-free extracts of strains bearing mutations in LAC4 by 2-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, lac4-14 or lac4-29 mutants. Of all lac4 mutants, only those bearing lac4-23 for lac4-30 contain a protein that cross-reacts with anti-.beta.-galactosidase antibody. .beta.-galactosidase activity in several Lac+ revertants of strains carrying lac4-23 or lac4-30 greatly decreased thermostability.