RESUMO
The occurrence of ovarian dysfunction is often due to the imbalance between the formation of reactive oxygen species (ROS) and the ineffectiveness of the antioxidative defense mechanisms. Primary sources of ROS are respiratory electron transfer and the activity of NADPH oxidases (NOX) while superoxide dismutases (SOD) are the main key regulators that control the levels of ROS and reactive nitrogen species intra- and extracellularly. Because of their central role SODs are the subject of research on human ovarian dysfunction but sample acquisition is low. The high degree of cellular and molecular similarity between Drosophila melanogaster ovaries and human ovaries provides this model organism with the best conditions for analyzing the role of ROS during ovarian function. In this study we clarify the localization of the ROS-producing enzyme dNox within the ovaries of Drosophila melanogaster and by a tissue-specific knockdown we show that dNox-derived ROS are involved in the chorion hardening process. Furthermore, we analyze the dSod3 localization and show that reduced activity of dSod3 impacts egg-laying behavior but not the chorion hardening process.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Ovário , Espécies Reativas de Oxigênio , Superóxido Dismutase , Animais , Drosophila melanogaster/genética , Feminino , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ovário/metabolismo , NADPH Oxidases/metabolismo , NADPH Oxidases/genética , Reprodução , NADPH Oxidase 5/metabolismo , NADPH Oxidase 5/genética , Oviposição , Córion/metabolismoRESUMO
The 57B region of Drosophila melanogaster includes a cluster of the three homeobox genes orthopedia (otp), Drosophila Retinal homeobox (DRx), and homeobrain (hbn). In an attempt to isolate mutants for these genes, we performed an EMS mutagenesis and isolated lethal mutants from the 57B region, among them mutants for otp, DRx, and hbn. With the help of two newly generated deletions from the 57B region, we mapped additional mutants to specific chromosomal intervals and identified several of these mutants from the 57B region molecularly. In addition, we generated mutants for CG15651 and RIC-3 by gene targeting and mutants for the genes CG9344, CG15649, CG15650, and ND-B14.7 using the CRISPR/Cas9 system. We determined the lethality period during development for most isolated mutants. In total, we analysed alleles from nine different genes from the 57B region of Drosophila, which could now be used to further explore the functions of the corresponding genes in the future.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Ligação a DNA/genética , Genes Homeobox , Proteínas ras/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismoRESUMO
Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.
Assuntos
Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Fosforilação/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Proteínas de Sinalização YAPRESUMO
The Drosophila homeodomain-interacting protein kinase (Hipk) is the fly representative of the well-conserved group of HIPKs in vertebrates. It was initially found through its characteristic interactions with homeodomain proteins. Hipk is involved in a variety of important developmental processes, such as the development of the eye or the nervous system. In the present study, we set Hipk and the Drosophila homeodomain proteins Homeobrain (Hbn), Empty spiracles (Ems), and Muscle segment homeobox (Msh) in an enzyme-substrate relationship. These homeoproteins are transcription factors that function during Drosophila neurogenesis and are, at least in part, conserved in vertebrates. We reveal a physical interaction between Hipk and the three homeodomain proteins in vivo using bimolecular fluorescence complementation (BiFC). In the course of in vitro phosphorylation analysis and subsequent mutational analysis we mapped several Hipk phosphorylation sites of Hbn, Ems, and Msh. The phosphorylation of Hbn, Ems, and Msh may provide further insight into the function of Hipk during development of the Drosophila nervous system.
