Inducible h-ras gene-expression controlled by an allosterically regulated transactivator

Inducible promoters have been employed widely to control the transcription of specific genes and thus to elucidate their role. We constructed a plasmid in which three lac operator sequences and the SV40 early promoter were placed upstream of the T24 H-ras1 gene. This plasmid was co-introduced in rat fibroblasts with a plasmid coding for a fusion protein containing the DNA binding domain of the lac repressor protein and the transactivation domain VP16 from HSV1. In the clones derived from this co-transfection, the transcription of the exogenous H-ras1 gene is dependent on the interaction between the transactivator protein and the lac operators. Given that this interaction is inhibited by IPTG, such a cell line overexpresses the H-ras gene encoded protein ras p21 and the addition of isopropyl beta-D-thiogalactopyranoside (IPTG) into the culture medium diminishes overexpression. The decrease of ms expression levels after the addition of IPTG was confirmed by Western blot analysis and by quantitative Reverse Transcription PCR based on comparison of amplification levels between the target cellular H-ras1 gene transcript and an in vitro produced deletion mutant H-ras reference transcript.