Cloning, structural analysis, and expression of the human slow twitch skeletal muscle/cardiac troponin C gene.
The two isoforms of troponin C that are differentially expressed in slow and fast twitch skeletal muscle are encoded by single copy genes. We are analyzing the mechanisms that control their highly restricted pattern of differential expression. The structure of the human fast twitch troponin C isoform gene has been reported (Gahlmann, R. and Kedes, L. (1990) J. Biol. Chem. 265, 12520-12528). Here we describe the isolation, nucleotide sequence, and localization of a regulating promoter element sufficient to impart expression of the human slow twitch skeletal muscle troponin C gene which is also the isoform expressed in heart. The 3.0-kilobase gene is composed of 6 exons and 5 introns. Introns and 5'-flanking sequences between the human and mouse slow troponin C genes are highly conserved. The gene is transcribed from the same start site in skeletal and cardiac muscle. A consensus TATA box is located 29 base pairs upstream of the transcriptional start site but no canonical CAAT box was observed. Cell transfection experiments provided evidence that promoter elements that are responsible for a cell type-specific pattern of gene expression are located in the 5'-flanking sequences. Constructs comprising 4.0 kilobases of 5'-flanking sequences, attached upstream of the chloramphenicol transferase gene as reporter, were expressed at high levels in differentiated cells of three myogenic cell lines (C2, L8, and H9c2(2-1)) and also at high levels in undifferentiated C2 and H9c2(2-1) cells. Chloramphenicol acetyltransferase activity was not detected in either WI38 cells or monkey kidney cells, CV-1. 5'-Deletion constructs were assayed for expression in differentiated H9c2(2-1) and C2 cells. Sequences between base pairs -67 and +24 were sufficient for high level expression in these cell lines.
Schreier
T
T
Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
Kedes
L
L
Gahlmann
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D015870:Gene Expression; D005796:Genes; D015698:Genomic Library; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D015345:Oligonucleotide Probes; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014336:Troponin / Q000235:genetics*; D019209:Troponin C
D015345:Oligonucleotide Probes; D014336:Troponin; D019209:Troponin C