Duchenne muscular dystrophy (DMD) is the most frequent of the childhood onset muscular dystrophies. Determination of the DMD gene(s) would allow the development of rational therapy. In addition, DNA restriction fragment length polymorphisms (RFLPs) closely linked to DMD gene(s) can be used in genetic counseling and prenatal diagnosis.
The specific aims would be to identify such probes of cloned DMD gene region (Xp2.1) or closely linked RFLPs. Identification and subsequent successful cloning of DMD gene could lead to the identification of the defective gene product and its role in functioning of the dystrophic muscle cell. DMD is an X-linked disease. Females with DMD are rare and, except those with XO configurations, have balanced X chromosome translocations with the breakpoint at Xp2.1. DNA from transformed lymphoid line (Fra 21) of one such rare female patient having a balanced chromosome translocation (X:21) (p2.1:p1.2) is available in our laboratory. Lambda phage libraries prepared from total genomic Fra 21 DNA and from bivariantly flow sorted X-derivative (X der) and 21 derivative (21 der) chromosomes will contain translocation junction or breakpoint clones. Since both the X der and 21 der stain at the breakpoint with silver, indicating the presence of ribosomal DNA (rDNA), cloned 18S and 28S DNA probes in plaque lift experiments will identify breakpoint clones. The rDNA clones containing breakpoint portions of X chromosome will be selected by in situ hybridization and by dot blot analysis of cell lines with variable number of X chromosomes. Unique DNA sequences from these clones will serve in RFLP segregation analysis of multigeneration DMD and Becker muscular dystrophy (BMD) families which are available to us. Unique sequence RFLPs, adjacent to and overlapping breakpoint sequences, will be generated using appropriate restriction enzymes. These will then be screened against human liver and skeletal muscle cDNA libraries (available in our lab) to define Xp2.1 specific clones. Using hybridization and translation methodologies, Xp2.1 specific clones can be utilized to detect differences in specific transcripts and protein products in normal and dystrophic muscle.
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