CLONING AND ANALYSIS OF THE PROMOTER REGION OF THE RAT SM22-ALPHA GENE

We have cloned and sequenced a 1.9 kb fragment of the 5'-upstream sequ ence of the smooth-muscle-specific gene SM22 alpha. The region cloned consisted of the SM22 alpha. promoter, a 65 bp exon containing most of the 5'-untranslated region and 307 bp of the first intron. A 1.5 kb f ragment at the 5' end of this sequence was able to drive the expressio n of a reporter chloramphenicol acetyltransferase (CAT) gene in both v ascular smooth-muscle cells and Rat-1 fibroblasts. This promoter regio n did not contain a consensus TATAA box but contained the sequence TTT AAA 25 bp from the major start site identified by primer extension. De letion analysis showed that a fragment of the promoter from +65 to -30 3 was more active in both cell types than the 1.5 kb fragment suggesti ng that there are silencer sequences in the region 5' to the core prom oter. CAT activity was also observed with fragments containing bases 65 to -193 and +65 to -117 in smooth-muscle cells. In contrast with th e smooth-muscle cells, no CAT activity was detected in Rat-1 fibroblas ts with the smallest two fragments. The residual promoter activity in the smallest fragment of the SM22 alpha promoter tested suggested that , unlike the smooth-muscle alpha-actin promoter, transcription from th e SM22 alpha promoter can occur in smooth-muscle cells in the absence of factors binding to CC(A/T-rich)(6)GG (CArG box) or CANNTG (E box) m otifs.