The goal of this proposal is to elucidate the mechanisms involved in the generation, by a single contractile protein gene, of multiple mRNAs encoding different proteins that are tissue-specific and developmentally regulated. This analysis will concentrate on the elucidation of the structure of the mammalian cardiac troponin T (TnT) gene and its regulation during development, in response to hormone stimulation and to work overload hypertrophy. It is not yet known whether this variety of stimuli are able to act on the gene in an independent manner, either directly or indirectly, or whether they all act through a common pathway affecting a unique DNA sequence in the gene. Moreover, it is not yet known whether the different genes expressed during development and in response to different stimuli have common regulatory sequences. The essential role of TnT in striated muscle contraction relates to its close interaction with tropomyosin and with the calcium binding protein troponin C in the sarcomere. Other striated muscle TnT genes utilize alternative splicing of common and isotype specific exons to generate isoform diversity in response to developmental and tissue-specific regulation by the production of multiple mRNAs. RNA splicing defects have been implicated as the mechanism contributing to several clinically relevant states (ie. Thalassemias). However, detailed mechanisms of alternative splicing remain obscure. This proposal will attempt to determine whether the mammalian cardiac TnT gene utilizes alternative splicing and to understand the mechanism(s) involved in this process. A three part approach will be followed. First, the cDNA and genomic clone corresponding to this gene will be isolated and characterized by differential colony hybridization, Northern blot hybridization, S1-nuclease analysis and DNA sequencing. Second, the normal and abnormal patterns of expression of this gene in the myocardium will be correlated in search for common features that could shed light on its mode of regulation. Analysis of these clones under different conditions will occur by S1-nuclease mapping and 5' extension techniques. Finally, the dissection of the molecular events involved in the regulation of this gene and of mechanisms of alternative splicing, if it occurs, will be initiated using a combination of in vivo and in vitro systems including specific gene constructs.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Clinical Investigator Award (CIA) (K08)
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Children's Hospital (Buffalo)
United States
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