1887

Abstract

Products of oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha- cDNA, together with pΔACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of pΔACMVCAT were also observed in cell lines carrying stably transfected oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha- expression increases the activity of a reporter gene, -galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha- (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha- (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by oncogenes.

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1995-04-01
2021-10-25
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