ABSTRACT
Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two dystrophin isoforms are expressed, Dp427 and Dp71. To characterize the function of Dp71 in myogenesis, we have examined the expression of Dp71 in myogenic cells. The localization of Dp71 in these cells is distinct from the localization of Dp427. Whereas Dp427 localizes to focal adhesions and surface membrane during myogenesis, Dp71 localizes to stress fiberlike structures in myogenic cells. Biochemical fractionation of myogenic cells demonstrates that Dp71 cosediments with the actin bundles thus confirming this interaction. Furthermore, transfection of C2C12 myoblasts with constructs encoding Dp71 fused to green fluorescent protein targeted the protein to the actin microfilament bundles. These results demonstrate involvement of Dp71 with the actin cytoskeleton during myogenesis and suggest a role for Dp71 that is distinct from Dp427.
Subject(s)
Dystrophin/analogs & derivatives , Dystrophin/physiology , Muscle, Skeletal/metabolism , Actins/metabolism , Animals , Blotting, Western , Cell Line , Cells, Cultured , Dystrophin/analysis , Dystrophin/genetics , Fetus , Gene Expression Regulation, Developmental , Green Fluorescent Proteins , Humans , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Microscopy, Fluorescence , Muscle, Skeletal/chemistry , Protein Isoforms , Recombinant Fusion Proteins , Reverse Transcriptase Polymerase Chain Reaction , Subcellular Fractions , TransfectionABSTRACT
Restriction fragment length polymorphism (RFLP) associated with the three human lenkocyte antigen (HLA)-D-region gene-specific cDNA probes (gene-specific DQ alpha, DQ beta and DP beta) was evaluated in two Caucasian families from southwestern Ontario, each with two confirmed narcoleptic siblings. The special feature of the two families is that the affected members are not necessarily DRw15 (previously DR2) positive. In family 1, of the two affected sibs one is DRw15 positive, whereas in family 2 all members including the two affected sibs are DRw15 negative. There is no association between narcolepsy and HLA haplotype in the two families. Further, the polymorphic DNA band patterns generated by a number of restriction enzymes do not show a relationship with the presence or absence of narcolepsy. The probe-specific DNA and patterns however do follow the HLA haplotypes associated with the DQ and DR loci. These results suggest that in DRw15-negative narcolepsy, the DNA patterns are not informative with respect to diagnosing or predicting the presence of narcolepsy. Further, such results argue for genetic heterogeneity in narcolepsy, and the role of DRw15 in the development of the disease could at best be viewed as contributory and not essential.