Immunochemical analysis of DMD gene product dystrophin

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The reseach was aimed to establish antibodies to varied domains of dystrophin and to analyze the physiological and pathogenetic significance of dystrophin.1) We synthesized three peptides of the amino acid sequences 215-264 (N terminal domain).10125-10138 and 10209-10229 (cyteine rich and C terminal domains). We succeeded to produce a hybridoma A1C secreting a monoclonal antibody IgG2a against the N terminal domain. However, We could not make antibodies to other domains.2) We analyzed the precise localization of dystrophin in myofibers. We could show a dense accumulation onto neuromuscular and myotendon junctions (Biomed.Res.10 ; 405-409.1989) as well as on sarcolemma. 3) At first, dystrophin was believed to be defective in DMD and either to be decreased in the amount or to be expressed in the abnormal size in BMD.However, we dimonstrated the pesence of revertant fibers in DMD which expressed the full size of dystrophin (Proc Japan Acad.ser.B 64 ; 205-208.1988). We succeeded in presenting various splicings of each BMD gene allele in 23 BMD patients (J.Neurol.Sci.121 ; 183-189,1994). 4) During myogenesis DRP was downregulated whereas dystrophin was upregulated during gestation. In various congenital myopathies dystrophin was well expressed although nucleus migration, mitochondrion maturity and myosin differentiation were variously disturbed. In congenital myotonic dystrophy, appearance of dystrophin was enormously delayd. The results confirmed the immaturity of the myofibers. 5) We isolated dystrophin from rabbit. The triton X extract of the heavy-membrane and myofibril fraction was sequentially processed in hydroxyapatite, WGA and DEAE column and we got 90% purity of dystrophin. The rotary shadowing demonstrated a dumbbell type rod. The size was -10nm thick and 3 nm wide. The result was in good accordance with the proposed model of antiparallel homodimer (Proc.Jap.Acad.Ser.B,66 ; 96-99,1990).