Koppe R I, Hallauer P L, Karpati G, Hastings K E
Montreal Neurological Institute, McGill University, Canada.
J Biol Chem. 1989 Aug 25;264(24):14327-33.
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.
我们已经对编码收缩调节蛋白肌钙蛋白I（快速型和慢速型骨骼肌亚型，即TnIfast和TnIslow）的啮齿动物mRNA的结构和表达进行了表征。从小鼠和大鼠肌肉cDNA克隆文库中分离出TnIfast和TnIslow cDNA克隆，并将其用作Northern印迹和原位杂交研究中的亚型特异性探针。这些研究表明，TnIfast和TnIslow mRNA在骨骼肌中表达，但在心肌或其他组织中不表达，并且它们在单个肌纤维中差异表达。基于TnI亚型mRNA含量的原位杂交分析进行的纤维分型与通过肌球蛋白ATP酶组织化学评估的纤维类型显示出极好的相关性。这些结果直接证明，脊椎动物横纹肌细胞各类别中骨骼肌TnI亚型的差异表达基于基因调控机制，该机制控制单个肌细胞中特定TnI mRNA的丰度。在大鼠L6和小鼠C2肌肉细胞系的分化培养物中，TnIfast和TnIslow mRNA均以相当的水平表达。因此，尽管神经元输入已被证明是决定骨骼肌中快速型与慢速型亚型特异性表达的重要因素，但在没有神经的情况下，TnIfast和TnIslow基因均可在肌细胞中表达。将推导的啮齿动物TnI氨基酸序列与先前确定的兔蛋白序列进行比较，结果表明，具有潜在快速/慢速亚型特异性功能的残基存在于几个离散的簇中，其中两个位于先前确定的肌动蛋白和肌钙蛋白C结合位点附近。
