Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells.
As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli beta-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid beta-galactosidase proteins. Conditions for transfection were varied to optimize the expression of beta-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli beta-galactosidase and distinct from the major lysosomal "acid" beta-galactosidase. The fused preproinsulin-beta-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric beta-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-beta-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed.
D000818:Animals; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007328:Insulin; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009876:Operon; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics*
D007328:Insulin; D011498:Protein Precursors; C013737:preproinsulin; D011384:Proinsulin; D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase