RESUMO
We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.
Assuntos
Actinas/metabolismo , Echinococcus/genética , Proteínas de Helminto/genética , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , DNA Complementar/química , Echinococcus/química , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , OvinosRESUMO
We have cloned the Drosophila gene discs degenerate-4 (dd4) and find that it encodes a component of the gamma-tubulin ring complex (gammaTuRC) homologous to Spc98 of budding yeast. This provides the first opportunity to study decreased function of a member of the gamma-tubulin ring complex, other than gamma-tubulin itself, in a metazoan cell. gamma-tubulin is no longer at the centrosomes but is dispersed throughout dd4 cells and yet bipolar metaphase spindles do form, although these have a dramatically decreased density of microtubules. Centrosomin (CNN) remains in broad discrete bodies but only at the focused poles of such spindles, whereas Asp (abnormal spindle protein) is always present at the presumptive minus ends of microtubules, whether or not they are focused. This is consistent with the proposed role of Asp in coordinating the nucleation of mitotic microtubule organizing centers. The centrosome associated protein CP190 is partially lost from the spindle poles in dd4 cells supporting a weak interaction with gamma-tubulin, and the displaced protein accumulates in the vicinity of chromosomes. Electron microscopy indicates not only that the poles of dd4 cells have irregular amounts of pericentriolar material, but also that they can have abnormal centrioles. In six dd4 cells subjected to serial sectioning centrioles were missing from one of the two poles. This suggests that in addition to its role in nucleating cytoplasmic and spindle microtubules, the gammaTuRC is also essential to the structure of centrioles and the separation of centrosomes.
Assuntos
Centrossomo/metabolismo , Proteínas de Drosophila , Drosophila/genética , Proteínas dos Microtúbulos/genética , Mutação , Tubulina (Proteína)/genética , Animais , Centrossomo/ultraestrutura , Cromossomos/metabolismo , Clonagem Molecular , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/ultraestrutura , Masculino , Metáfase/genética , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Subunidades Proteicas , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101(lcd). To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIbeta. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.
