The 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene contains evolutionarily conserved sequence motifs within a functional promoter.
The 5'-flanking, 5'-untranslated, and amino-terminal protein coding regions of the single-copy 13-kilobase mouse vascular smooth muscle (VSM) alpha-actin gene have been cloned and sequenced. Respectively, there is 73 and 89% homology from the start of transcription (+1) to a point 206 base pairs upstream when comparing mouse to chicken and mouse to human VSM alpha-actin 5'-flanking region sequences. Two proximal 16-base pair motifs containing putative cis-acting regulatory elements having the configuration CC(A/T)6GG were found to be 100% conserved and present in the same position upstream from the transcription start site in all three species. A third more distal CC(A/T)6GG-like motif was 100% conserved between only the mouse and human genes whereas a fourth motif was unique to the mouse gene. The two upstream motifs may be important in controlling VSM alpha-actin gene transcription in mammals. Cell transfection assays using hGH reporter gene fusion plasmids showed that all four CC(A/T)6GG elements were required for tissue-specific, core promoter activity and were able to direct hGH expression in both mouse BC3H1 myogenic cells and early-passage rabbit aortic smooth muscle cells. The core promoter was not active in mouse fibroblasts suggesting that the region between -372 and -143 may mediate tissue-restrictive expression of the VSM alpha-actin gene. A putative "cell density responsive element" may be located between -1074 and -372 since fusion plasmids containing this portion of the VSM alpha-actin 5'-flanking region were significantly more active in promoting hGH expression in inducible, density-activated BC3H1 myoblasts compared to aortic smooth muscle cells which are largely constitutive for VSM alpha-actin expression.
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Ohio State Biochemistry Program, Ohio State University, Wooster 44691.
Foster
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Strauch
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D000199:Actins / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D002460:Cell Line; D005796:Genes; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D000199:Actins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone