|Budget Amount *help
¥6,800,000 (Direct Cost : ¥6,800,000)
Fiscal Year 1996 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1995 : ¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1994 : ¥2,300,000 (Direct Cost : ¥2,300,000)
C-protein is a major myosin-binding protein involved in myosin assembly and registration of thick filaments in the sarcomeres during myofibrillogenesis. Distinct C-protein isoforms, fast (F), slow (S), and cardiac (C), are present in both chicken and mammals, and C-isoform is known to be first expressed during chicken muscle development. The present studies were carried out aiming to clarify the molecular structure of C-protein and its role in myofibrillogensis, and the following results were obtained.
1) The complete cDNA sequence encoding chicken cardiac (C) C-protein and the deduced amino acid sequence were determined. Seven IgC2 domains, 3 fibronectin type III domains and a phosphorylation site were specified in the molecule and the C-terminal IgC2 was designated to be essential for myosin-binding. In addition, two variants of C isoform which differ only in the presence or absence of the phosphorylation site were detected. Both were detected in cardiac muscle through developmental s
tages, while only the latter was in embryonic skeletal muscle.
2) In order to clarify expression pattern of C-protein isoforms and their roles in mammalian muscle development, the cDNAs encoding mouse skeletal muscle (F and S) C-protein were cloned and determined the entire sequences. In adult mouse tissues, the messages for C-, F-, S- isoforms were detected in a tissue-specific fasion by Northern blotting ; interestingly, however, both F- and S-isoforms were detectable in EDL (extensor digitorum longus) muscle which has been categorized as a typical fast muscle. Although C-isoform is known to be expressed first and transiently during development of chicken skeletal muscles, C-isoform was not detected at all in mouse skeletal muscle through developmental stages, but S-isoform became detectable first, followed by appearance of F-isoform. Basically the same results were obtained at a protein level by means of Western blot and indirect immunofluorescence methods with isoform-specific antibodies. Less