Myogenic determination factors are basic helix-loop-helix proteins that govern specification and differentiation of muscle cells, and bind to the E-box consensus sequence CANNTG in promoter regions of Show more
Myogenic determination factors are basic helix-loop-helix proteins that govern specification and differentiation of muscle cells, and bind to the E-box consensus sequence CANNTG in promoter regions of muscle-specific genes. No E-box mutation has been reported to date. RAPSN encodes rapsyn, a 43 kDa postsynaptic peripheral membrane protein that clusters the nicotinic acetylcholine receptor at the motor endplate. Transcriptional regulation mechanisms of RAPSN have not been studied. We here report two novel E-box mutations in the RAPSN promoter region in eight congenital myasthenic syndrome patients. Patient 1 carries -27C-->G that changes an E-box at -27 to -22 from CAGCTG to GAGCTG. An allele harboring -27C-->G is not transcribed in patient's muscle. Patients 2-8 are of Oriental Jewish stock of Iraqi or Iranian origin with facial malformations, and harbor -38A-->G that changes another E-box at -40 to -35 from CAACTG to CAGCTG, which does not affect the consensus CANNTG sequence. Haplotype analysis shows that -38A-->G arises from a common founder. For each mutation, position +1 represents the major transcriptional start site that we determine to be 172 nucleotides upstream of the translational start site. Electrophoretic mobility shift assays reveal that -38A-->G gains, and -27C-->G looses, binding affinity for different components of nuclear extracts of C2C12 myotubes. Luciferase reporter assays show that both -38A-->G and -27C-->G attenuate reporter gene expression in C2C12 myotubes, and that -27C-->G additionally attenuates reporter gene expression in MyoD- or myogenin-transfected HEK cells. The -27C-->G mutation also markedly attenuates the enhancer activity of an E-box on an SV40 promoter. Impaired transcriptional activities of the RAPSN promoter region predict reduced rapsyn expression and endplate acetylcholine receptor deficiency. Show less
The rat dilute-opisthotonus (dop) autosomal recessive gene, causing ataxia and coat color dilution, was mapped on chromosome 8 by PCR-amplified microsatellite markers. To facilitate the linkage analys Show more
The rat dilute-opisthotonus (dop) autosomal recessive gene, causing ataxia and coat color dilution, was mapped on chromosome 8 by PCR-amplified microsatellite markers. To facilitate the linkage analysis, an intersubspecific cross with a Japanese wild rat strain was used. The recombination frequencies were 12.8% between Apoc3 and dop, and 32.1% between dop and Mylc1v. The following order of three genes is proposed; Apoc3-dop-Mylc1v. This mutation appears to be homologous to dilute-lethal (d1) of the mouse in terms of clinical symptoms, coat color effect and chromosomal location of the gene loci. Key words: ataxic mutant rat, dilute-opisthotonus (dop), gene mapping. Show less