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Abstract
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.
Highlights
Clone RCTlO proved to be a full length clone functional comparison, Tobacman andLee (1987) have demcontaining 50 base pairs of 5’-untranslated se- onstrated that these two isoforms have different influences quence, 870 bp of coding sequence, and 196 bp plus on the overallresponse of reconstitutedthinfilamentsto poly(A) tail at the 3“untranslatedregion. In the other Ca2+.These results raise thpeossibility that regulationof the cDNA clone (RCTll), the entire 3”untranslated se- expression of these two isoforms may modulate myocardial quence andmost of the coding sequencewere identical function
Present in the coding region from residues 18 to 27. terminal region by the replacementof 7 glutamic acid residues. This insertion sequence appeared to code for a frag- by neutral amino acids, two forms appear to have ment (EDWSEEEEDE)highly enriched in acidic amintohe identical numbeorf amino acid residues
We describe the isolation and characterization of TnT cDNA clones from young rat cardiac muscle
Summary
The restriction endonucleases and othermodifying enzymes used in this studwy ere purchased from New England Biolabs except where another vendor is indicated. Buffer containing 0.4 M NaCI, 1 mM EDTA, 40 mM PIPES, pH 6.4, and 80% formamide. S1 nuclease (100 units, Pharmacia LKB Biotechnology, Inc.) in 400 pl of buffer containing 50 mM sodium acetate, pH 4.6, 1 mM ZnClz, 250 mM NaCI, 50 pg/mI bovine serum albumin was added to thehybridization mixture and incubateda t 22 "C for 30 min. (A)' RNA Preparation-In order to construct a rat cardiac EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol),heatedto cDNA library that contained clones representing boTthn T isoforms, 70 "C for 10 min, and loaded immediately onto 4-8% polyacrylamide, poly (A)' RNA was isolated from 4.5 g of day 5 rat hearts and 1.5 g 8 M urea sequencing gels. (A)' RNA was separated end-labeled A-DNA fragments. Isolated poly(A)+ RNA was examined by in vitro translationanalysis usinga rabbit reticulocyte translationsystem
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