RESUMO
valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , RNA Helicases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Quinase do Ponto de Checagem 2 , Clonagem Molecular , RNA Helicases DEAD-box , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Humanos , Dados de Sequência Molecular , Mutação/genética , Oogênese , Ovário/citologia , Ovário/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.
