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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL038868-01
Application #
3355304
Study Section
Biochemistry Study Section (BIO)
Project Start
1987-07-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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Fattal, P G; Billadello, J J (1993) Species-specific differential cleavage and polyadenylation of plasminogen activator inhibitor type 1 hnRNA. Nucleic Acids Res 21:1463-6
Hawker, K J; Billadello, J J (1993) A multiplex polymerase chain reaction colony miniprep. Biotechniques 14:764-5
Fattal, P G; Schneider, D J; Sobel, B E et al. (1992) Post-transcriptional regulation of expression of plasminogen activator inhibitor type 1 mRNA by insulin and insulin-like growth factor 1. J Biol Chem 267:12412-5
Trask, R V; Koster, J C; Ritchie, M E et al. (1992) The human M creatine kinase gene enhancer contains multiple functional interacting domains. Nucleic Acids Res 20:2313-20
Hopkins, W E; Westerhausen Jr, D R; Sobel, B E et al. (1991) Transcriptional regulation of plasminogen activator inhibitor type-1 mRNA in Hep G2 cells by epidermal growth factor. Nucleic Acids Res 19:163-8
Fontanet, H L; Trask, R V; Haas, R C et al. (1991) Regulation of expression of M, B, and mitochondrial creatine kinase mRNAs in the left ventricle after pressure overload in rats. Circ Res 68:1007-12
Ritchie, M E; Trask, R V; Fontanet, H L et al. (1991) Multiple positive and negative elements regulate human brain creatine kinase gene expression. Nucleic Acids Res 19:6231-40
Westerhausen Jr, D R; Hopkins, W E; Billadello, J J (1991) Multiple transforming growth factor-beta-inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in Hep G2 cells. J Biol Chem 266:1092-100
Trask, R V; Billadello, J J (1990) Tissue-specific distribution and developmental regulation of M and B creatine kinase mRNAs. Biochim Biophys Acta 1049:182-8

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