Jin J P, Lin J J
Department of Biology, University of Iowa, Iowa City 52242.
J Biol Chem. 1989 Aug 25;264(24):14471-7.
We have previously reported that troponin T (TnT), a regulatory protein in muscle contraction, undergoes a switch from the larger, acidic embryonic form to the smaller, basic adult form during rat heart development (Jin, J.-P., and Lin, J.J.-C. (1988) J. Biol. Chem. 263, 7309-7315). To investigate the significance and the molecular mechanism of this isoform switching, cDNA clones encoding rat cardiac TnT were obtained by screening a cDNA library constructed from young rat cardiac poly(A)+ RNAs using the expression vector lambda gt11. Clone RCT10 proved to be a full length clone containing 50 base pairs (bp) of 5'-untranslated sequence, 870 bp of coding sequence, and 196 bp plus poly(A) tail at the 3'-untranslated region. In the other cDNA clone (RCT11), the entire 3'-untranslated sequence and most of the coding sequence were identical with that of RCT10, but an additional 30-bp insert was present in the coding region from residues 18 to 27. This insertion sequence appeared to code for a fragment (EDWSEEEEDE) highly enriched in acidic amino acid residues. Thus, RCT11 might represent a clone encoding the embryonic isoform of rat cardiac TnT. S1 nuclease RNA mapping analysis using end-labeled RCT11 cDNA probes confirmed that this region of the sequence is different in embryonic and adult isoform mRNAs. These results suggest that both embryonic and adult isoforms of rat cardiac TnT are generated from the same primary transcript by developmentally regulated alternative splicing. The amino acid sequence deduced from RCT10 cDNA exhibits 87%, 85%, and 72% homology with bovine, rabbit, and chicken cardiac TnTs, respectively, but less homology (57-59%) with the known skeletal TnTs from human, rat, rabbit, and chicken. Moreover, both the 5'- and the 3'-untranslated sequences of rat cardiac TnT mRNA are completely different from those reported for rat skeletal TnT mRNA, suggesting that rat cardiac TnT is coded from a gene distinct from the rat skeletal TnT gene.
我们先前曾报道，肌钙蛋白T（TnT）是一种肌肉收缩中的调节蛋白，在大鼠心脏发育过程中会从较大的酸性胚胎形式转变为较小的碱性成年形式（Jin, J.-P., and Lin, J.J.-C. (1988) J. Biol. Chem. 263, 7309 - 7315）。为了研究这种同工型转换的意义和分子机制，通过使用表达载体λgt11筛选由幼鼠心脏多聚腺苷酸加尾RNA构建的cDNA文库，获得了编码大鼠心脏TnT的cDNA克隆。克隆RCT10被证明是一个全长克隆，在5'-非翻译序列中有50个碱基对（bp），编码序列有870 bp，在3'-非翻译区有196 bp加上多聚腺苷酸尾巴。在另一个cDNA克隆（RCT11）中，整个3'-非翻译序列和大部分编码序列与RCT10相同，但在编码区从第18位到第27位有一个额外的30-bp插入片段。这个插入序列似乎编码一个在酸性氨基酸残基上高度富集的片段（EDWSEEEEDE）。因此，RCT11可能代表一个编码大鼠心脏TnT胚胎同工型的克隆。使用末端标记的RCT11 cDNA探针进行的S1核酸酶RNA图谱分析证实，该序列区域在胚胎和成年同工型mRNA中是不同的。这些结果表明，大鼠心脏TnT的胚胎和成年同工型都是由发育调控的可变剪接从同一初级转录本产生的。从RCT10 cDNA推导的氨基酸序列与牛、兔和鸡的心脏TnT分别具有87%、85%和72%的同源性，但与已知的人、大鼠、兔和鸡的骨骼肌TnT的同源性较低（57 - 59%）。此外，大鼠心脏TnT mRNA的5'-和3'-非翻译序列与报道
