RESUMO
Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates; innexins/pannexins encode gap-junction proteins in prechordates and chordates. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely. The phenomenon of rectification was first described five decades ago, but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal). When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.
Assuntos
Drosophila/fisiologia , Sinapses Elétricas/fisiologia , Animais , Animais Geneticamente Modificados , Conexinas/genética , Conexinas/fisiologia , Drosophila/anatomia & histologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Fenômenos Eletrofisiológicos , Feminino , Marcação de Genes , Genes de Insetos , Ativação do Canal Iônico , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Oócitos/metabolismo , Fenótipo , Terminações Pré-Sinápticas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , XenopusRESUMO
Locus control regions (LCRs) are defined by their ability to confer reproducible physiological levels of transgene expression in mice and therefore thought to possess the ability to generate dominantly a transcriptionally active chromatin structure. We report the first characterization of a muscle-cell-specific LCR, which is linked to the human desmin gene (DES). The DES LCR consists of five regions of muscle-specific DNase I hypersensitivity (HS) localized between -9 and -18 kb 5' of DES and reproducibly drives full physiological levels of expression in all muscle cell types. The DES LCR DNase I HS regions are highly conserved between humans and other mammals and can potentially bind a broad range of muscle-specific and ubiquitous transcription factors. Bioinformatics and direct molecular analysis show that the DES locus consists of three muscle-specific (DES) or muscle preferentially expressed genes (APEG1 and SPEG, the human orthologue of murine striated-muscle-specific serine/threonine protein kinase, Speg). The DES LCR may therefore regulate expression of SPEG and APEG1 as well as DES.
Assuntos
Desmina/genética , Região de Controle de Locus Gênico , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Sequência Conservada , DNA/genética , DNA/metabolismo , Desoxirribonuclease I , Expressão Gênica , Ligação Genética , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculos/metabolismo , Proteínas Serina-Treonina Quinases , Ratos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Transcrição GênicaRESUMO
Splicing and 3'-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3'-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human beta-globin gene is necessary to stimulate mRNA 3'-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring beta-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3'-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3'-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3'-end processing, which contributes to 3'-terminal exon definition.
