RESUMO
BK-type calcium-activated potassium channels are large conductance channels that respond to changes in intracellular calcium and membrane potential. These channels are used in a wide variety of cell types and have recently been linked to drug sensitivity and tolerance. In both Drosophila and mammals, BK channels are encoded by the slowpoke gene. The Drosophila slowpoke gene includes 14 alternative exons distributed among five sites of alternative splicing. Presumably, the purpose of alternative processing is to provide transcripts tailored to the needs of the cell. The slowpoke gene is expressed in nervous, muscle and epithelial tissues. To determine whether splicing is controlled in a tissue- and/or developmental-specific manner, we built tissue- and developmental-specific cDNA libraries that preserved the relative frequency of various slowpoke splice variants. These libraries were screened by colony hybridization using alternative exon-specific DNA probes to document the frequency of individual alternative exons in different developmental stages and distinct tissue types. We demonstrate that slowpoke transcripts undergo tissue- and developmental-specific splicing in Drosophila and some exons are diagnostic for specific tissues.
Assuntos
Processamento Alternativo/genética , Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Animais , Sistema Digestório/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Éxons/genética , Amplificação de Genes/genética , Biblioteca Gênica , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Larva/genética , Músculos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos/genéticaRESUMO
In this paper we describe the isolation of a cDNA that encodes a human TFIIAalpha/beta-like factor (ALF). The open reading frame of ALF predicts a protein of 478 amino acids that contains characteristic N- and C-terminal conserved domains separated by an internal nonconserved domain. In addition, a rare ALF-containing cDNA, which possesses an extended N terminus (Stoned B/TFIIAalpha/beta-like factor; SALF) has also been identified. The results of Northern and dot blot analyses show that ALF is expressed almost exclusively in testis; in contrast, TFIIAalpha/beta and TFIIAgamma are enriched in testis but are also widely expressed in other human tissues. Recombinant ALF (69 kDa) and TFIIAgamma (12 kDa) polypeptides produced in Escherichia coli form an ALF/gamma complex that can stabilize TBP-TATA interactions in an electrophoretic mobility shift assay. The ALF/gamma complex is also able to restore transcription from the adenovirus major late promoter using HeLa cell nuclear extracts that have been depleted of TFIIA. Overall, the data show that ALF is a functional homolog of human general transcription factor TFIIAalpha/beta that may be uniquely important to testis biology.
