RESUMO
Translin is an evolutionarily conserved approximately 27-kDa protein that binds to specific DNA and RNA sequences and has diverse cellular functions. Here, we report the cloning and characterization of the translin orthologue from the fruit fly Drosophila melanogaster. Under protein-denaturing conditions, purified Drosophila translin exists as a mixture of dimers and monomers just like human translin. In contrast to human translin, the Drosophila translin dimers do not appear to be stabilized by disulfide interactions. Drosophila translin shows a ubiquitous cytoplasmic localization in early embryonal syncytial stage, with an enhanced staining in ventral neuroblasts at later stages (8-9), which are probably at metaphase. An elevated expression was seen in several other cell types, such as cells around the tracheal pits in the embryo and oenocytes in the third instar larva. RNA in situ hybridization showed an increased expression in the ventral midline cells of the larval brain, suggesting a neuronal expression, which was corroborated by protein immunostaining. In adult flies, Drosophila translin is localized in the brain neuronal cell bodies and in early spermatocytes. Interestingly, Drosophila translin mutants exhibit an impaired motor response which is sex specific. Taken together, the multiple cellular localizations, the high neuronal expression and the attendant locomotor defect of the Drosophila translin mutant suggest that Drosophila translin may have roles in neuronal development and behavior analogous to that of mouse translin.
Assuntos
Comportamento Animal/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Mutação/genética , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Clonagem Molecular , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Éxons/genética , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Larva , Dados de Sequência Molecular , Atividade Motora , Proteínas Mutantes/metabolismo , Fenótipo , Estrutura Quaternária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Trax, expressed alone aggregates into insoluble complexes, whereas upon co-expression with Translin becomes readily soluble and forms a stable heteromeric complex ( approximately 430 kDa) containing both proteins at nearly equimolar ratio. Based on the subunit molecular weights, estimated by MALDI-TOF-MS, the purified complex appears to comprise of either an octameric Translin plus a hexameric Trax (calculated MW 420 kDa) or a heptamer each of Trax and Translin (calculated MW 425 kDa) or a hexameric Translin plus an octameric Trax (calculated MW 431 kDa). The complex binds single-stranded/double-stranded DNA. ssDNA gel-shifted complex shows both proteins at nearly equimolar ratio, suggesting that Translin "chaperones" Trax and forms heteromeric complex that is DNA binding competent.