Cytochemical observation of regulated bacterial beta-galactosidase gene expression in mammalian cells.
Bacterial beta-galactosidase, encoded by the lacZ gene, serves as a sensitive cytochemical marker in eukaryotic cells and tissues. In transient expression experiments, human and simian cells stain blue 48 hr after transfection with a plasmid containing a lacZ gene, whose expression is directed by a simian virus 40 promoter containing a synthetic lactose operator sequence. Transfection efficiency was about 0.6%. Incorporation of an operator sequence within the promoter permits regulation of beta-galactosidase gene expression by the lacI gene product, the lac repressor. When cells were cotransfected with the lacZ plasmid and a second plasmid containing the lacI gene, beta-galactosidase activity was extinguished. Its activity could be reestablished to original levels upon application of isopropyl beta-D-thiogalactoside to transfected cells. A cell line that stably carries both the lacI and lacZ genes was efficiently induced to synthesize beta-galactosidase after isopropyl beta-D-thiogalactoside administration. In transient expression experiments and in stably transfected lines, repression and induction of beta-galactosidase activity were predominantly at the transcriptional level.
Liu
H S
HS
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, OH 45267-0521.
Feliciano
E S
ES
Stambrook
P J
PJ
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D004790:Enzyme Induction; D004794:Enzyme Repression; D005696:Galactosidases / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006801:Humans; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D012329:RNA, Bacterial; D005696:Galactosidases; D001616:beta-Galactosidase