The genes encoding isoenzymes of creatine kinase (CK) represent a multigene family whose members are expressed in a developmentally-regulated and tissue-specific fashion. The CK gene family will serve as a model system for investigating the molecular mechanisms underlying the selective induction of genes during development. In addition, characterization of the post- translational modifications of isoenzymes of CK released into the circulation may be useful for the very early and specific diagnosis of myocardial infarction. The overall objectives of the proposed research are to elucidate the structure of the creatine kinase gene family, the regulation of tissue specific expression of the creatine kinase genes at selected stages of development under normal physiologic conditions, in pathologic states, with differentiation of muscle cells in culture, and with respect to the post-translational modification of isoenzymes of creatine kinase in the circulation. We will characterize the biochemical changes which result in the post-translational modification of MB creatine kinase by plasma factors to isoforms and the time course of conversion of isoforms of MB CK In the circulation after myocardial infarction. We will determine the tissue specific expression of the creatine kinase genes by probing Northern blots and dot blots of total cytoplasmic RNA, purified from tissues, with cDNA clones which we have developed and have shown to be specific for M and B CK mRNAs. We will measure steady state levels of mRNA encoding M and B CK in brain, heart, and skeletal muscle from rats at selected stages of development and in rat hearts undergoing hypertrophy in response to aortic banding by probing Northern blots with M and B CK specific cDNA probes. We will isolate and determine the structure and organization of the human genes encoding M, B, and mitochondrial creatine kinase. We will determine whether specific DNA sequences of the human genes encoding M and B creatine kinase confer regulation of their expression during development by transfecting differentiating BC3H1 cells in culture with chimeric plasmids containing 5' upstream sequences of human CK genes fused with the bacterial chloramphenicol acetyltransferase gene in a transient expression system.
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