Abstract
We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.
Highlights
Afg AGGIle Thr AlaArg Arg Gln H i s Leu Lys Si; Val Met Leu Gln I l e Ala Ala ATC ACG GCC CGG AGA CAGCAC CTG AAG AGT GTG ATG CTC CAG ATA GCG GCC mRNAs are expressedin cultured muscle cell lines in the
Probes in Northern blot anind situhybridization stud- (TnI)’ prevents the contractile interactionof actin and myoies
Neuronal input has been shown The expression of T n I isoforms has been studied by bioto be an important factor in determining fast versus chemical andimmunohistochemicalmethods indeveloping slow isoform-specific expressioninskeletal muscle, and adult muscle [4, 10, 11]and under conditions of experiboth TnIf, and TnIslOwgenes can be expressed imnus- mentaldenervation [12] andcross-reinnervation [13, 14]
Summary
Ile Thr AlaArg Arg Gln H i s Leu Lys Si; Val Met Leu Gln I l e Ala Ala ATC ACG GCC CGG AGA CAGCAC CTG AAG AGT GTG ATG CTC CAG ATA GCG GCC mRNAs are expressedin cultured muscle cell lines in the. HIS Cys Pro Pro Leu HIS ILE Pro Gly Ser Met SER G1U V a l Gln Glu Leu CYSLys Gln CAC TGC CCG CCA CTG CAC ATC CCA GGC TCC ATG TCT GAA GTG CAG GAA CTC TGCAAA CAA a comparative analysis of TnI amino acid sequences which identifies clusters of putative fast uersus slow isoform-specific residueslocatedin or near previouslyidentified TnC and actin binding sites on theT n l molecule. 50% formamide, 42 "C, with washes in 0.1 X SSC, 65 "C) with portion of the cM113 cDNA insert (the first nucleotide of cM113 oligo(dT)-primed 32P-labeledcDNA made on poly(A)+RNA extracted corresponding to TnIe., mRNA is the C in codon 8: the 3'-end EcoRI from predominantlyfast(gastrocnemius) and predominantly slow site of cM113 isindicated in lower case letters at the end of the (soleus) rat muscles, to identify slow muscle-enriched sequences. High stringencyhybridizationconditions chain termination method [20] using the United States Biochemical (see above) were used
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