Cloning and analysis of the promoter region of the rat SM22α gene

Abstract

We have cloned and sequenced a 1.9 kb fragment of the 5'-upstream sequence 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 fragment at the 5' end of this sequence was able to drive the expression of a reporter chloramphenicol acetyltransferase (CAT) gene in both vascular smooth-muscle cells and Rat-1 fibroblasts. This promoter region did not contain a consensus TATAA box but contained the sequence TTTAAA 25 bp from the major start site identified by primer extension. Deletion analysis showed that a fragment of the promoter from +65 to -303 was more active in both cell types than the 1.5 kb fragment suggesting that there are silencer sequences in the region 5' to the core promoter. CAT activity was also observed with fragments containing bases +65 to -193 and +65 to -117 in smooth-muscle cells. In contrast with the smooth-muscle cells, no CAT activity was detected in Rat-1 fibroblasts 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 the SM22 alpha promoter can occur in smooth-muscle cells in the absence of factors binding to CC(A/Trich)6GG (CArG box) or CANNTG (E box) motifs.

Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity

“…Cytoplasmic RNA was isolated from cells in culture as described previously [32]. RNA (1 µg) was reverse transcribed using avian myelomablastosis virus reverse transcriptase in a 20-µl reaction as described previously [12]. The cDNA produced by these reactions (2 µl) was amplified by PCR using Taq DNA polymerase as described previously [12] [33] and are shown in the 5h 3h orientation).…”

“…RNA (1 µg) was reverse transcribed using avian myelomablastosis virus reverse transcriptase in a 20-µl reaction as described previously [12]. The cDNA produced by these reactions (2 µl) was amplified by PCR using Taq DNA polymerase as described previously [12] [33] and are shown in the 5h 3h orientation). Primers A and B were designed to amplify the full coding sequence for SRF, primer C was designed to amplify from the end of exon 1, thus avoiding the GC-rich region present in exon 1 which reduces the efficiency of the PCR.…”

“…The orientation of the clones was determined by restriction digestion with PstI and the sequence of clones in the correct orientation was confirmed. Northern-blot analysis was carried out using α-tropomyosin, SM22α and β-actin cDNA probes as described previously [12,34,35].…”

“…promoter respectively [12], and pSVβ-gal. Cell lysates were prepared 48 h after transfection and assayed for CAT and β-galactosidase activity.…”

“…Recently, however, it has been found that SRF also plays a major role in the control of muscle gene expression and muscle differentiation. For example, the promoters that drive the expression of smoothmuscle myosin heavy chain, smooth-muscle α-actin and SM22α all contain multiple CArG [CC(A\Trich)6GG] boxes, the binding site for SRF [9][10][11][12][13]. These CArG boxes have been shown to be important in the control of gene expression in smooth-muscle cells in itro.…”