Kinetics of expression of inducible beta-galactosidase in murine fibroblasts: high initial rate compared to steady-state expression.
We have constructed a murine fibroblast cell line in which synthesis of beta-galactosidase can be induced by incubation with isopropyl-beta-D-thiogalactopyranoside (IPTG). This was obtained by transfection by both a plasmid expressing lacI and a second plasmid expressing lacZ from a modified simian virus 40 (SV40) promoter containing a lac operator. We have measured the induction kinetics as well as the basal and induced differential rate of synthesis of beta-galactosidase. The steady-state rate of synthesis is tenfold higher in the presence than in the absence of inducer; we calculate an average of 1200 lacZ polypeptides are synthesized per minute per cell in the induced cultures. However, immediately after induction, the rate of accumulation of beta-galactosidase is up to 50-fold higher than the basal level. Based on our measurements of stability of beta-galactosidase, we suggest that induction may result in a subsequent down-modulation of the transcriptional activity from the induced gene. We hypothesize this inhibition may result from structural changes in DNA components, such as nucleosomes.
Jacobsen
K D
KD
Department of Molecular Cell Biology, University of Copenhagen, Denmark.
Willumsen
B M
BM
D000818:Animals; D002460:Cell Line; D004790:Enzyme Induction; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D015870:Gene Expression; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D013539:Simian virus 40; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis*
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase