Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Sep 15;310(Pt 3):1037–1043. doi: 10.1042/bj3101037

Cloning and analysis of the promoter region of the rat SM22 alpha gene.

P R Kemp 1, J K Osbourn 1, D J Grainger 1, J C Metcalfe 1
PMCID: PMC1135999  PMID: 7575400

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.

Full text

PDF
1041

Images in this article

1039Figure 1
on p.1039
1040Figure 3
on p.1040
1041Figure 5
on p.1041
1042Figure 7
on p.1042

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blank R. S., McQuinn T. C., Yin K. C., Thompson M. M., Takeyasu K., Schwartz R. J., Owens G. K. Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation. J Biol Chem. 1992 Jan 15;267(2):984–989. [PubMed] [Google Scholar]
  2. Boxer L. M., Miwa T., Gustafson T. A., Kedes L. Identification and characterization of a factor that binds to two human sarcomeric actin promoters. J Biol Chem. 1989 Jan 15;264(2):1284–1292. [PubMed] [Google Scholar]
  3. Boxer L. M., Prywes R., Roeder R. G., Kedes L. The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor. Mol Cell Biol. 1989 Feb;9(2):515–522. doi: 10.1128/mcb.9.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carroll S. L., Bergsma D. J., Schwartz R. J. Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene. An actin gene which produces multiple messenger RNAs. J Biol Chem. 1986 Jul 5;261(19):8965–8976. [PubMed] [Google Scholar]
  5. Duband J. L., Gimona M., Scatena M., Sartore S., Small J. V. Calponin and SM 22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development. Differentiation. 1993 Dec;55(1):1–11. doi: 10.1111/j.1432-0436.1993.tb00027.x. [DOI] [PubMed] [Google Scholar]
  6. Ernø H., Monard D. Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter. Gene Expr. 1993;3(2):163–174. [PMC free article] [PubMed] [Google Scholar]
  7. Grainger D. J., Hesketh T. R., Metcalfe J. C., Weissberg P. L. A large accumulation of non-muscle myosin occurs at first entry into M phase in rat vascular smooth-muscle cells. Biochem J. 1991 Jul 1;277(Pt 1):145–151. doi: 10.1042/bj2770145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Katoh Y., Loukianov E., Kopras E., Zilberman A., Periasamy M. Identification of functional promoter elements in the rabbit smooth muscle myosin heavy chain gene. J Biol Chem. 1994 Dec 2;269(48):30538–30545. [PubMed] [Google Scholar]
  9. Kemp P. R., Grainger D. J., Shanahan C. M., Weissberg P. L., Metcalfe J. C. The Id gene is activated by serum but is not required for de-differentiation in rat vascular smooth muscle cells. Biochem J. 1991 Jul 1;277(Pt 1):285–288. doi: 10.1042/bj2770285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kemp P. R., Shachar-Hill Y., Weissberg P. L., Metcalfe J. C. Inhibition of PDGF BB stimulated DNA synthesis in rat aortic vascular smooth muscle cells by the expression of a truncated PDGF beta receptor. FEBS Lett. 1993 Dec 20;336(1):119–123. doi: 10.1016/0014-5793(93)81623-8. [DOI] [PubMed] [Google Scholar]
  11. Kim J. H., Johansen F. E., Robertson N., Catino J. J., Prywes R., Kumar C. C. Suppression of Ras transformation by serum response factor. J Biol Chem. 1994 May 13;269(19):13740–13743. [PubMed] [Google Scholar]
  12. Kobayashi R., Kubota T., Hidaka H. Purification, characterization, and partial sequence analysis of a new 25-kDa actin-binding protein from bovine aorta: a SM22 homolog. Biochem Biophys Res Commun. 1994 Feb 15;198(3):1275–1280. doi: 10.1006/bbrc.1994.1180. [DOI] [PubMed] [Google Scholar]
  13. Lassar A. B., Buskin J. N., Lockshon D., Davis R. L., Apone S., Hauschka S. D., Weintraub H. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell. 1989 Sep 8;58(5):823–831. doi: 10.1016/0092-8674(89)90935-5. [DOI] [PubMed] [Google Scholar]
  14. Lees-Miller J. P., Heeley D. H., Smillie L. B. An abundant and novel protein of 22 kDa (SM22) is widely distributed in smooth muscles. Purification from bovine aorta. Biochem J. 1987 Jun 15;244(3):705–709. doi: 10.1042/bj2440705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mar J. H., Ordahl C. P. M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol Cell Biol. 1990 Aug;10(8):4271–4283. doi: 10.1128/mcb.10.8.4271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Min B. H., Foster D. N., Strauch A. R. The 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene contains evolutionarily conserved sequence motifs within a functional promoter. J Biol Chem. 1990 Sep 25;265(27):16667–16675. [PubMed] [Google Scholar]
  17. Nishida W., Kitami Y., Abe M., Hiwada K. Gene cloning and nucleotide sequence of SM22 alpha from the chicken gizzard smooth muscle. Biochem Int. 1991 Mar;23(4):663–668. [PubMed] [Google Scholar]
  18. Nishida W., Kitami Y., Hiwada K. cDNA cloning and mRNA expression of calponin and SM22 in rat aorta smooth muscle cells. Gene. 1993 Aug 25;130(2):297–302. doi: 10.1016/0378-1119(93)90435-6. [DOI] [PubMed] [Google Scholar]
  19. Osbourn J. K., Weissberg P. L., Shanahan C. M. A regulatory element downstream of the rat SM22 alpha gene transcription start point enhances reporter gene expression in vascular smooth muscle cells. Gene. 1995 Mar 10;154(2):249–253. doi: 10.1016/0378-1119(94)00847-l. [DOI] [PubMed] [Google Scholar]
  20. Pfeifer G. P., Tanguay R. L., Steigerwald S. D., Riggs A. D. In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev. 1990 Aug;4(8):1277–1287. doi: 10.1101/gad.4.8.1277. [DOI] [PubMed] [Google Scholar]
  21. Prinjha R. K., Shapland C. E., Hsuan J. J., Totty N. F., Mason I. J., Lawson D. Cloning and sequencing of cDNAs encoding the actin cross-linking protein transgelin defines a new family of actin-associated proteins. Cell Motil Cytoskeleton. 1994;28(3):243–255. doi: 10.1002/cm.970280307. [DOI] [PubMed] [Google Scholar]
  22. Reddy S., Ozgur K., Lu M., Chang W., Mohan S. R., Kumar C. C., Ruley H. E. Structure of the human smooth muscle alpha-actin gene. Analysis of a cDNA and 5' upstream region. J Biol Chem. 1990 Jan 25;265(3):1683–1687. [PubMed] [Google Scholar]
  23. Shanahan C. M., Weissberg P. L., Metcalfe J. C. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ Res. 1993 Jul;73(1):193–204. doi: 10.1161/01.res.73.1.193. [DOI] [PubMed] [Google Scholar]
  24. Takahashi K., Nadal-Ginard B. Molecular cloning and sequence analysis of smooth muscle calponin. J Biol Chem. 1991 Jul 15;266(20):13284–13288. [PubMed] [Google Scholar]
  25. Thweatt R., Lumpkin C. K., Jr, Goldstein S. A novel gene encoding a smooth muscle protein is overexpressed in senescent human fibroblasts. Biochem Biophys Res Commun. 1992 Aug 31;187(1):1–7. doi: 10.1016/s0006-291x(05)81449-4. [DOI] [PubMed] [Google Scholar]
  26. Weintraub H., Davis R., Tapscott S., Thayer M., Krause M., Benezra R., Blackwell T. K., Turner D., Rupp R., Hollenberg S. The myoD gene family: nodal point during specification of the muscle cell lineage. Science. 1991 Feb 15;251(4995):761–766. doi: 10.1126/science.1846704. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES