TRANSCRIPTIONAL REGULATORY AND RESPONSE MAPPING OF THE RAT HA-RAS UPSTREAM SEQUENCE USING PRIMARY MAMMARY EPITHELIAL-CELLS

The rat Ha-ras upstream sequence (-2876 to +986 bp relative to the most 5' transcriptional start site) was transcriptionally mapped art a gross level. Ha-ras upstream sequence and 5' unidirectional deletion reporter constructs were transfected via particle bombardment into primary cultures of rat mammary epithelial cells. Analyses of Ha-ras reporter expression show that a fragment extending from -2876 to -2110 bp contains a positive regulatory sequence. The majority of Ha-ras expression is attributed to this sequence, since its deletion results in a 4-fold decrease in expression. The Ha-ras gene was also assessed for autoregulation using similar co-transfection experiments. Wild-type and activated (codon 12 G-->A transition) Ha-ras expression vectors, transcriptionally driven by Ha-ras upstream sequence, were co-transfected with Ha-ras upstream sequence deletion reporter constructs. The activated Ha-ras gene product induced its own transcription 2-fold, targeting the regulatory region between -2119 and -313 bp.