Kinetics of Expression of Inducible β-Galactosidase in Murine Fibroblasts: High Initial Rate Compared to Steady-state Expression

Abstract

We have constructed a murine fibroblast cell line in which synthesis of β-galactosidase can be induced by incubation with isopropyl-β- d- thiogalactopyranoside (IPTG). This was obtained by transfection by both a plasmid expressing lacIand a second plasmid expressing lacZfrom a modified simian virus 40 (SV40) promoter containing a lacoperator. We have measured the induction kinetics as well as the basal and induced differential rate of synthesis of β-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 lacZpolypeptides are synthesized per minute per cell in the induced cultures. However, immediately after induction, the rate of accumulation of β-galactosidase is up to 50-fold higher than the basal level. Based on our measurements of stability of β-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.