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The muscleblind-like (MBNL) proteins are tissue-specific alternative splicing regulators. Dysfunction of MBNL has been implicated in the pathogenesis of expanded CUG repeats-associated myotonic dystrophy (DM). In this study, we describe the identification and functional characterization of a Caenorhabditis elegans muscleblind (CeMbl) gene. CeMbl is a single gene alternatively spliced to generate two isoforms (CeMBL-A and CeMBL-B). It displays a high homology with human MBNL1 in the C3H1 zinc finger domains. CeMbl transcripts are detected in larval and adult stages. However, inactivation of CeMbl by RNA-mediated interference results in muscle phenotype only at adulthood. Immunofluorescence staining using anti-vinculin antibody reveals that the organization of dense body is disrupted in affected worms. Our results demonstrate a growth-dependent requirement of CeMbl on muscle structure and function. They also provide a possible molecular basis for the developmentally regulated toxicity of expanded CUG repeats.

p73 is one of the p53 family members which are transcription factors involved in the regulation of cell proliferation, apoptosis, and differentiation. In this study, we cloned the p73 cDNA from zebrafish ovary RNA. The consensus open reading frame (1923 bp) encodes a polypeptide of 640 amino acids which shares 70–95% identity to the p73 of other vertebrates. Expression of zebrafish p73 mRNA is restricted to tissues such as skin, fin, brain, ovary, and testis, in contrast to the ubiquitous expression of zebrafish p53 and p63. During embryonic development, p73 transcripts are detected from the zygote period to the early larva stage. Whole-mount in situ hybridization reveals that p73 expression is in the brain, including olfactory bulbs, telencephalon, and hypothalamus, as well as in the pharyngeal arches and the nose. Moreover, p73 protein is found in the ovary and testis sections by immunohistochemical staining.

In this study, we investigated the effect of NO donor, diethylamine/nitric oxide (DEA/NO), on the electrophysiological behavior of human skeletal muscle chloride channel (CLCN1). The wild-type and variants of CLCN1, including one polymorphism (P727L) and four mutants (T631I, D644G, G482R, and S471F), were expressed in Xenopus oocytes and the ionic current was measured by two-electrode voltage-clamp method. Our results revealed that there is no significant difference in the current-voltage relationships and half-voltage values of open probability between wild-type and variants of CLCN1 except for G482R. Application of the DEA-NO (0.1mM) significantly increases the channel conductance of wild-type, T631I, D644G, and S471F, but not P727L. This indicates that P727L polymorphism causes loss of sensitivity of CLCN1 to the DEA/NO treatment, which could be due to a conformational change caused by proline substitution. The data suggest that the polymorphic changes may affect the function of CLCN1 in response to the treatment of chemical compounds.

Expansion of CTG repeat within the 3'-untranslated region of the DMPK gene causes the most common neuromuscular disorder, myotonic dystrophy type 1 (DM1), through a RNA trans-dominant mechanism. Here, we explore Caenorhabditis elegans as a model system to investigate the repeat size-dependent toxic effect by expression of green fluorescent protein (GFP) transcripts with various lengths of untranslatable CUG repeats (CUG5, CUG30, CUG83, CUG125, and CUG213) in body wall muscles. CUG213 animals died during embryogenesis or showed retarded growth at larval stages due to defective muscle development. CUG125 animals, although can produce offspring, exhibited uncoordinated muscle function, deviated electropharyngeogram, and an age-dependent abnormality in muscle structure. Most CUG83 animals had normal muscle structure and function as those expressing 30 and shorter repeats. Our results demonstrate for the first time that the in vivo toxicity of CUG repeats is repeat length- and growth-regulated and suggest that expanded CUG repeats are sufficient to cause congenital-like phenotypes in living organisms.

Mutations in the CLCN1 gene frequently associate with myotonia congenita (MC). We have recently reported several CLCN1 mutants in Taiwanese patients. To further elucidate the correlation between the genotypes and phenotypes, in this study, we used Xenopus oocyte as a system to investigate the functional effects of these mutants. The fs793X and G482R mutants, which were suggested to have a dual inheritance pattern, were found to cause a functional loss of CLCN1 channels. While co-expression of fs793X and wild-type (WT) showed a reduction of chloride conductance by about half of WT channels, the activation curve of voltage-dependence was not shifted. A compound heterozygous mutant, P575S/D644G, was found in a patient. When both mutants were co-expressed in oocytes, they caused a shift of the voltage-dependence of activation curve to more positive values than individual mutant. This indicates that both P575S and D644G mutants may contribute cooperatively to change the gating property of CLCN1 channel. Interestingly, the S471F mutant did not cause significant alternation of functional properties. Consistent with the fact that T631I mutant was found in three asymptomatic individuals, the electrophysiological parameters of T631I were similar to those of WT CLCN1 channels, suggesting that T631I is a neutral mutation. These results further clarify the correlation between the mutations and their functional implications of CLCN1 channels.