Abstract
The contractile protein troponin I is encoded by a multigene family whose members are expressed differentially in various classes of muscle fibers. In vertebrates, the "slow" isoform of troponin I is expressed during early heart and skeletal muscle development but is restricted to slow twitch skeletal muscle in the adult. This diverse expression pattern offers an opportunity to study the regulation of a single gene within different developmental contexts. To initiate such studies, we have cloned the gene encoding the human slow twitch skeletal muscle isoform of troponin I and have identified 5'-flanking sequences required for its expression in skeletal muscle cells. The slow troponin I gene spans 12.5 kilobases and is divided into nine exons. In contrast to many muscle-specific genes, the troponin I promoter does not contain consensus CCAAT or TATA elements. Moreover, the sequence from -9 to +11 resembles an "initiator element" previously shown to direct transcription of some tissue-specific genes lacking TATA boxes (Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113; Brand, N. J., Petkovich, M., and Chambon, P. (1990) Nucleic Acids Res. 18, 6799-6806; Weis, L., and Reinberg, D. (1992) FASEB J. 6, 3300-3309). A transcriptional fusion construct, comprising 4.2 kilobases of troponin I 5'-flanking DNA linked to the bacterial chloramphenicol acetyl-transferase gene, exhibited cell type-specific and developmentally regulated expression. A muscle-specific enhancer regulated slow troponin I promoter activity.
Highlights
In contrast to many muscle-specific genes, no conopmentally regulated expression.A muscle-specific en- sensus TATA nor CCAAT boxes were found within the approhancer regulated slow troponinI promoter activity
Organization of the Human troponin I (TnI), Gene-A human genomic library was screened with a TnI, cDNA probe
Promoter Sequence a n d Dunscription Znitiation within the Human TnZ, Gene-In a previous report we described a cDNA that was a full-length representative of a human TnI, mRNA [11].Primer extension analysis presented in that report indicated the likely presence of two transcription start sites, one to this are12 nucleotides complementary tothe region of the TnI, gene immediatelyupstream from the 5' transcriptioninitiationsite.The defined by the 5' end of the full-length cDNA, and the other site3'-most 15 nucleotides of the probe comprise the noncomplementary
Summary
Cloning and Structural Characterization of the Human TnZ, Gene-A human genomic library was constructed by partial digestion of human lymphocyteDNAwith the restriction endonucleaseMboI. To determine intron sizes of the human TnI, gene, the exon-specific For each transfection, a totalof 10pg of plasmid DNA (7 pg of TnI,CAT primers were used in PCR amplifications of TnI,genomic and plasmid plus 3 pg of pRSVZ) was used. As a size marker for myocyte-specific RNAprotection products; the TnI, The 4-kb insert of this genomic construct extended from the vector- cRNA was generated by T7 promoter-initiated transcription of the TnI, derived Sal siteof phage clone 7Dto the BamHI site within intron 4of cDNA cloned into pGEM-3 (Riboprobe GeminiSystem, Promega). B’-untranslated sequence was isolated from the genomic phage clone 21C (Fig. 1)and, after the addition of HindIII linkers, was cloned into the HindIII site of the CAT expression vector pHCAOCAT [16] This initial promoter construct was designated -4200Tn1,CAT.
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