Assuntos
Proteínas de Drosophila/metabolismo , Células Germinativas/fisiologia , Glicoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Animais , Divisão Celular , Núcleo Celular/metabolismo , Senescência Celular , Drosophila/citologia , Drosophila/embriologia , Drosophila/fisiologia , Células Germinativas/citologia , Células Germinativas/metabolismo , Ligantes , Masculino , Fosforilação , Transdução de Sinais , Espermátides/citologia , Espermátides/fisiologia , Espermatogênese , Células-Tronco/citologia , Células-Tronco/metabolismo , Testículo/citologiaRESUMO
Dorsoventral polarity in the Drosophila embryo is established through a signal transduction cascade triggered in ventral and ventrolateral regions. Activation of a transmembrane receptor, Toll, leads to localized recruitment of the adaptor protein Tube and protein kinase Pelle. Signaling through these components directs degradation of the IkappaB-like inhibitor Cactus and nuclear translocation of the Rel protein Dorsal. Here we show through confocal immunofluorescence microscopy that Pelle functions to downregulate the signal-dependent relocalization of Tube. Inactivation of the Pelle kinase domain, or elimination of the Tube-Pelle interaction, dramatically increases Tube recruitment to the ventral plasma membrane in regions of active signaling. We also characterize a large collection of pelle alleles, identifying the molecular lesions in these alleles and their effects on Pelle autophosphorylation, Tube phosphorylation and Tube relocalization. Our results point to a mechanism operating to modulate the domain or duration of signaling downstream from Tube and Pelle.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas de Insetos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular , Alelos , Sequência de Aminoácidos , Animais , Padronização Corporal , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Retroalimentação , Genes de Insetos , Proteínas de Insetos/genética , Mamíferos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Receptores Toll-LikeRESUMO
A number of fundamental processes comprise the cell division cycle, including spindle formation, chromosome segregation, and cytokinesis. Our current understanding of these processes has benefited from the isolation and analysis of mutants, with the meiotic divisions in the male germline of Drosophila being particularly well suited to the identification of the required genes. We show here that the fumble (fbl) gene is required for cell division in Drosophila. We find that dividing cells in fbl-deficient testes exhibit abnormalities in bipolar spindle organization, chromosome segregation, and contractile ring formation. Cytological analysis of larval neuroblasts from null mutants reveals a reduced mitotic index and the presence of polyploid cells. Molecular analysis demonstrates that fbl encodes three protein isoforms, all of which contain a domain with high similarity to the pantothenate kinases of A. nidulans and mouse. The largest Fumble isoform is dispersed in the cytoplasm during interphase, concentrates around the spindle at metaphase, and localizes to the spindle midbody at telophase. During early embryonic development, the protein localizes to areas of membrane deposition and/or rearrangement, such as the metaphase and cellularization furrows. Given the role of pantothenate kinase in production of Coenzyme A and in phospholipid biosynthesis, this pattern of localization is suggestive of a role for fbl in membrane synthesis. We propose that abnormalities in synthesis and redistribution of membranous structures during the cell division cycle underlie the cell division defects in fbl mutant cells.
Assuntos
Divisão Celular/genética , Drosophila melanogaster/genética , Meiose/genética , Mitose/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Clonagem Molecular , Coenzima A/biossíntese , Citoplasma/metabolismo , DNA Complementar/metabolismo , Embrião não Mamífero/metabolismo , Immunoblotting , Masculino , Camundongos , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Mutação , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Mapeamento Físico do Cromossomo , Isoformas de Proteínas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Testículo/metabolismo , Fatores de TempoRESUMO
Experiments reported in the past year have revealed considerable diversity in Toll-mediated pathways for signal transduction in development and innate immunity. Rather than function as a well conserved signaling cassette, Toll receptors and associated factors have apparently evolved as a diverse set of configurations to defend against microbial infection in species ranging from plants to humans.
Assuntos
Proteínas de Drosophila , Proteínas de Insetos/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos , Transdução de Sinais , Animais , Drosophila/genética , Drosophila/fisiologia , Humanos , Proteínas de Insetos/genética , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular/genética , Receptor 5 Toll-Like , Receptores Toll-LikeRESUMO
The fruitless gene governs courtship in male, but not female, Drosophila, yet it is expressed and specifically spliced in the brains of both sexes. New experiments reveal that a splice-recognition site retained in the mature message in females provides the basis for sex-specific translational repression.
Assuntos
Corte , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Biossíntese de Proteínas/genética , Caracteres Sexuais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Processamento Alternativo/genética , Animais , Feminino , Masculino , Músculos/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribonucleoproteínas/metabolismoRESUMO
A member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family was identified in Drosophila. DTRAF1 contains 7 zinc finger domains followed by a TRAF domain, similar to mammalian TRAFs and other members of the family identified in data bases from Caenorhabditis elegans, Arabidopsis, and Dictyostelium. Analysis of DTRAF1 binding to different members of the human TNF receptor family showed that this protein can interact through its TRAF domain with the p75 neurotrophin receptor and weakly with the lymphotoxin-beta receptor. DTRAF1 can also self-associate and binds to human TRAF1, TRAF2, and TRAF4. Interestingly, DTRAF1 interacts with human cIAP-1 and cIAP-2 but not with Drosophila DIAP-1 and -2. By itself, DTRAF1 did not induce significant NFkappaB activation when overexpressed in mammalian cells, although it specifically increased NFkappaB induction by TRAF6. In contrast, TRAF2-mediated NFkappaB induction was partially inhibited by DTRAF1. Mutants of DTRAF1 lacking the N-terminal region inhibited NFkappaB induction by either TRAF2 or TRAF6. DTRAF1 specifically associated with the regulatory N-terminal domain of Pelle, a Drosophila homolog of the human kinase interleukin-1 receptor-associated kinase (IRAK). Interestingly, though Pelle and DTRAF1 individually were unable to induce NFkappaB in a human cell line, co-expression of Pelle and DTRAF1 resulted in significant NFkappaB activity. Interactions of DTRAF1 with human TRAF-, TNF receptor-, and IAP-family proteins imply strong evolutionary conservation of TRAF protein structure and function throughout Metazoan evolution.
Assuntos
Proteínas de Drosophila , Drosophila/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis , Caenorhabditis elegans , Dictyostelium , Humanos , Quinases Associadas a Receptores de Interleucina-1 , Dados de Sequência Molecular , Ligação Proteica , Proteínas Quinases/metabolismo , Receptores de Interleucina-1/metabolismo , Fator 1 Associado a Receptor de TNF , Fator 2 Associado a Receptor de TNF , Fator 4 Associado a Receptor de TNF , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral , Dedos de ZincoRESUMO
The Drosophila Formin Homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, we generated embryos deficient for Diaphanous and analyzed three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. Our findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, we propose that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle.
Assuntos
Proteínas de Transporte/genética , Divisão Celular/genética , Proteínas de Drosophila , Drosophila/embriologia , Proteínas dos Microfilamentos , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Membrana Celular/metabolismo , Proteínas Contráteis/metabolismo , Citoesqueleto/metabolismo , Drosophila/genética , Forminas , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Proteínas de Insetos/metabolismo , Metáfase/genética , Mutação , Miosinas/metabolismo , Fenótipo , Transdução de SinaisRESUMO
The interaction of the serine/threonine kinase Pelle and adaptor protein Tube through their N-terminal death domains leads to the nuclear translocation of the transcription factor Dorsal and activation of zygotic patterning genes during Drosophila embryogenesis. Crystal structure of the Pelle and Tube death domain heterodimer reveals that the two death domains adopt a six-helix bundle fold and are arranged in an open-ended linear array with plastic interfaces mediating their interactions. The Tube death domain has an insertion between helices 2 and 3, and a C-terminal tail making significant and indispensable contacts in the heterodimer. In vivo assays of Pelle and Tube mutants confirmed that the integrity of the major heterodimer interface is critical to the activity of these molecules.
Assuntos
Morte Celular , Proteínas de Drosophila , Proteínas de Insetos/química , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Fatores de Transcrição , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia , Dimerização , Proteínas de Insetos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Boule, a Drosophila orthologue of the vertebrate Dazl fertility factors, is a testis-specific regulator of meiotic entry and germline differentiation. Mutations inactivating either Boule, which is an RNA-binding protein, or Twine, which is a Cdc25-type phosphatase, block meiotic entry in males. Here we show that twine and boule interact genetically. We also find that protein expression from twine messenger RNA correlates with cytoplasmic accumulation of Boule and is markedly reduced by boule mutations. Remarkably, heterologous expression of Twine rescues the boule meiotic-entry defect, indicating that the essential function of Boule at the transition from G2 to M phase during meiosis is in the control of Twine translation.
Assuntos
Proteínas de Drosophila , Proteínas de Insetos/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatócitos/citologia , Animais , Ciclo Celular , Centrossomo/fisiologia , Centrossomo/ultraestrutura , Proteína 1 Suprimida em Azoospermia , Drosophila/fisiologia , Masculino , Meiose , Oligospermia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Ligação a RNA/genética , Testículo/fisiologia , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Stimulation of the type 1 IL-1R (IL-1R1) and the IL-18R by their cognate ligands induces recruitment of the IL-1R-associated kinase (IRAK). Activation of IRAK leads in turn to nuclear translocation of NF-kappaB, which directs expression of innate and adaptive immune response genes. To study IRAK function in cytokine signaling, we generated cells and mice lacking the IRAK protein. IRAK-deficient fibroblasts show diminished activation of NF-kappaB when stimulated with IL-1. Immune effector cells without IRAK exhibit a defective IFN-gamma response to costimulation with IL-18. Furthermore, mice lacking the Irak gene demonstrate an attenuated response to injected IL-1. Deletion of Irak, however, does not affect the ability of mice to develop delayed-type hypersensitivity or clear infection with the intracellular parasite, Listeria monocytogenes. These results demonstrate that although IRAK participates in IL-1 and IL-18 signal transduction, residual cytokine responsiveness operates through an IRAK-independent pathway.
Assuntos
Citocinas/fisiologia , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/imunologia , Animais , Linhagem Celular , Feminino , Fertilidade/genética , Fertilidade/imunologia , Hipersensibilidade Tardia/genética , Hipersensibilidade Tardia/imunologia , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/fisiopatologia , Interleucina-1/antagonistas & inibidores , Interleucina-1/fisiologia , Quinases Associadas a Receptores de Interleucina-1 , Interleucina-18/fisiologia , Listeriose/genética , Listeriose/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Deleção de Sequência , Transdução de Sinais/genética , Baço/imunologia , Células-Tronco , Análise de SobrevidaRESUMO
A signaling pathway initiated by activation of the transmembrane receptor Toll defines dorsoventral polarity in the Drosophila embryo. Toll, which is present over the entire surface of the embryo, is activated ventrally by interaction with a spatially restricted, extracellular ligand. Tube and Pelle then transduce the signal from activated Toll to a complex of Dorsal and Cactus. Here we demonstrate by an mRNA microinjection assay that targeting of either Tube or Pelle to the plasma membrane by myristylation is sufficient to activate the signal transduction pathway that leads to Dorsal nuclear translocation. Using confocal immunofluorescence microscopy we also show that activated Toll induces a localized recruitment of Tube and Pelle to the plasma membrane. Together, these results strongly support the hypothesis that intracellular signaling requires the Toll-mediated formation of a membrane-associated complex containing both Tube and Pelle.
Assuntos
Padronização Corporal , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Embrião não Mamífero/fisiologia , Proteínas de Insetos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Animais Geneticamente Modificados , Membrana Celular/metabolismo , Embrião não Mamífero/citologia , Proteínas de Insetos/biossíntese , Microinjeções , Ácido Mirístico/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
We have identified a localized RNA component of Xenopus germ plasm. This RNA, Xdazl (Xenopus DAZ-like), encodes a protein homologous to human DAZ (Deleted in Azoospermia), vertebrate DAZL and Drosophila Boule proteins. Human males deficient in DAZ have few or no sperm and boule mutant flies exhibit complete azoospermia and male sterility. Xdazl RNA was detected in the mitochondrial cloud and vegetal cortex of oocytes. In early embryos, the RNA was localized exclusively in the germ plasm. Consistent with other organisms, Xdazl RNA was also expressed in the spermatogonia and spermatocytes of frog testis. Proteins in the DAZ-family contain a conserved RNP domain implying an RNA-binding function. We have shown that Xdazl can function in vitro as an RNA-binding protein. To determine if the function of Xdazl in spermatogenesis was conserved, we introduced the Xdazl cDNA into boule flies. This resulted in rescue of the boule meiotic entry phenotype, including formation of spindles, phosphorylation of histone H3 and completion of meiotic cell division. Overall, these results suggest that Xdazl may be important for primordial germ cell specification in the early embryo and may play a role analogous to Boule in promoting meiotic cell division.
Assuntos
Proteínas de Drosophila , Meiose/genética , Oócitos/química , RNA Mensageiro/análise , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Citoplasma/química , Proteína 1 Suprimida em Azoospermia , Drosophila , Genes , Humanos , Masculino , Mitocôndrias/química , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espermatogênese/genética , Espermatogônias/química , Espermatozoides/química , Testículo , XenopusRESUMO
The Drosophila boule gene is expressed exclusively in the male germline and encodes an RNA binding protein closely related to the mammalian fertility factors encoded by the DAZ (Deleted in Azoospermia) and DAZL (DAZ-like) genes. Mutation of boule blocks both meiotic divisions. Differentiation nonetheless continues, resulting in tetraploid spermatids that fail to mature into sperm. We have found that Boule localizes premeiotically to a perinucleolar region and then translocates to the cytoplasm at the onset of meiosis. We show that deletion of the Y chromosome ks-1 fertility locus eliminates Boule nuclear localization, although it does not perturb entry into meiosis. Based on these observations we propose that Boule acts in the cytoplasm to regulate the stability or translation of messenger RNA encoding an essential meiotic factor.
Assuntos
Proteínas de Drosophila , Drosophila/fisiologia , Proteínas de Ligação a RNA/análise , Espermatogênese , Animais , Proteína 1 Suprimida em Azoospermia , Drosophila/citologia , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Proteínas de Ligação a RNA/fisiologiaRESUMO
OBJECTIVE: To identify behaviors that indicate anxiety during a gynecologic examination. STUDY DESIGN: Five hundred twenty-two women visiting a private obstetrician/gynecologist's office completed the A-State scale of the State-Trait Anxiety Inventory and specific questions about their first pelvic examination and experiences with health practitioners performing subsequent gynecologic examinations. In addition, the hand placement a woman exhibited as the speculum was inserted was recorded, as were the reasons for her visit, reports of any symptoms, performance of any special procedures (e.g., colposcopy) and whether the pelvic examination was her first. RESULTS: Five behaviors observed during speculum insertion--holding hands/eyes covered or shut, hands on shoulders, hands covering pelvis, hands on legs, hands holding table--indicated increased anxiety. Together these behaviors were exhibited by one of every four patients and were found to be associated with high levels of anxiety. Greater anxiety was related to colposcopy, a less positive first pelvic examination experience, overall less positive experiences with examiners and performance of the first gynecologic examination at the present visit. CONCLUSION: Easily recognizable behaviors reflecting high anxiety in gynecologic patients were identified. Upon recognizing these behaviors, examiners can take necessary measures to reduce patient anxiety and prevent delays in and avoidance of gynecologic examinations.
Assuntos
Ansiedade , Comportamento , Genitália Feminina , Ginecologia , Exame Físico/psicologia , Adaptação Psicológica , Adolescente , Adulto , Fatores Etários , Idoso , Criança , Feminino , Doenças dos Genitais Femininos , Humanos , Pessoa de Meia-Idade , Grupos RaciaisRESUMO
A signaling pathway active on the ventral side of the Drosophila embryo defines dorsoventral polarity. A cell surface signal relayed by Toll, Tube and Pelle releases the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus; free Dorsal translocates into nuclei and directs expression of ventral fates. Using the yeast two-hybrid system and immunoprecipitation experiments, we define scaffolding and anchoring interactions among the pathway components. We show that Dorsal binds specifically to Tube, Pelle and Cactus, and that the protein kinase activity of Pelle differentially regulates its interactions with Dorsal and Tube. We also identify Drosophila Filamin as a potential adaptor linking the interaction network, via Tube, to the transmembrane receptor Toll.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Drosophila , Drosophila/embriologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animais , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Clonagem Molecular , Citoplasma/fisiologia , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/fisiologia , Genes Reporter , Proteínas de Insetos/metabolismo , Modelos Biológicos , Proteínas Nucleares/biossíntese , Fosfoproteínas/biossíntese , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Transdução de Sinais , Fatores de Transcrição/metabolismo , beta-Galactosidase/biossínteseRESUMO
The end-to-end association of chromosomes through their telomeres has been observed in normal cells of certain organisms, as well as in senescent and tumor cells. The molecular mechanisms underlying this phenomenon are currently unknown. We show here that five independent mutant alleles in the Drosophila UbcD1 gene cause frequent telomere-telomere attachments during both mitosis and male meiosis that are not seen in wild type. These telomeric associations involve all the telomeres of the D. melanogaster chromosome complement, albeit with different frequencies. The pattern of telomeric associations observed in UbcD1 mutants suggests strongly that the interphase chromosomes of wild-type larval brain cells maintain a Rab1 orientation within the nucleus, with the telomeres and centromeres segregated to opposite sides of the nucleus. The UbcD1 gene encodes a class I ubiquitin-conjugating (E2) enzyme. This indicates that ubiquitin-mediated proteolysis is normally needed to ensure proper telomere behavior during Drosophila cell division. We therefore suggest that at least one of the targets of UbcD1 ubiquitination is a telomere-associated polypeptide that may help maintain proper chromosomal orientation during interphase.
Assuntos
Aberrações Cromossômicas , Drosophila melanogaster/genética , Genes de Insetos , Telômero/genética , Animais , Sequência de Bases , Encéfalo/ultraestrutura , Drosophila melanogaster/enzimologia , Feminino , Teste de Complementação Genética , Hibridização in Situ Fluorescente , Masculino , Meiose/genética , Mitose/genética , Dados de Sequência Molecular , Mutação , Mapeamento por Restrição , Análise de Sequência de DNA , Fatores SexuaisRESUMO
Regulated proteolysis of Cactus, the cytoplasmic inhibitor of the Rel-related transcription factor Dorsal, is an essential step in patterning of the Drosophila embryo. Signal-induced Cactus degradation frees Dorsal for nuclear translocation on the ventral and lateral sides of the embryo, establishing zones of gene expression along the dorsoventral axis. Cactus stability is regulated by amino-terminal serine residues necessary for signal responsiveness, as well as by a carboxy-terminal PEST domain. We have identified Drosophila casein kinase II (CKII) as a Cactus kinase and shown that CKII specifically phosphorylates a set of serine residues within the Cactus PEST domain. These serines are phosphorylated in vivo and are required for wild-type Cactus activity. Conversion of these serines to alanine or glutamic acid residues differentially affects the levels and activity of Cactus in embryos, but does not inhibit the binding of Cactus to Dorsal. Taken together, these data indicate that wild-type axis formation requires CKII-catalyzed phosphorylation of the Cactus PEST domain.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila/embriologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sítios de Ligação , Padronização Corporal , Caseína Quinase II , Embrião não Mamífero/metabolismo , Ácido Glutâmico , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Serina/metabolismo , Transdução de SinaisRESUMO
Sexual orientation and courtship behavior in Drosophila are regulated by fruitless (fru), the first gene in a branch of the sex-determination hierarchy functioning specifically in the central nervous system (CNS). The phenotypes of new fru mutants encompass nearly all aspects of male sexual behavior. Alternative splicing of fru transcripts produces sex-specific proteins belonging to the BTB-ZF family of transcriptional regulators. The sex-specific fru products are produced in only about 500 of the 10(5) neurons that comprise the CNS. The properties of neurons expressing these fru products suggest that fru specifies the fates or activities of neurons that carry out higher order control functions to elicit and coordinate the activities comprising male courtship behavior.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Genes de Insetos/fisiologia , Proteínas do Tecido Nervoso/genética , Diferenciação Sexual/genética , Comportamento Sexual Animal/fisiologia , Fatores de Transcrição/genética , Fatores Etários , Animais , Sequência de Bases , Sistema Nervoso Central/fisiologia , Clonagem Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Dados de Sequência Molecular , Splicing de RNA/fisiologia , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genética , Dedos de Zinco/genéticaRESUMO
Dorsoventral polarity in the Drosophila embryo is established by a signaling pathway active on the ventral and ventrolateral surfaces of the embryo. Signal transduction via the protein kinase Pelle frees the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus, allowing Dorsal to translocate into ventral and ventrolateral nuclei and direct gene expression. Here, we show by immunochemical analyses that Pelle-mediated signaling induces the spatially graded degradation of Cactus. Using a tissue culture system which reconstitutes Pelle-dependent Cactus degradation, we show that a motif in Cactus resembling the sites of signal-dependent phosphorylation in the vertebrate homologs IkappaB-alpha and IkappaB-beta is essential for Pelle-induced Cactus degradation. Substitution of four serines within this motif with nonphosphorylatable alanine residues generated a mutant Cactus that still functions as a Dorsal inhibitor but is resistant to induced degradation. Injection of RNA encoding this altered form of Cactus has a dominant negative effect on establishment of dorsoventral polarity in the embryo. We conclude that dorsoventral signaling results in a Cactus concentration gradient and propose that signal-dependent phosphorylation directs the spatially regulated proteolysis of Cactus protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Embrião não Mamífero/fisiologia , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteínas de Ligação a DNA/química , Embrião não Mamífero/citologia , Imuno-Histoquímica , Dados de Sequência Molecular , Fosfoproteínas/química , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Infertility resulting from a severe defect in sperm production affects 2% of men worldwide. Of these men with azoospermia, the absence of sperm in semen, one in eight carry de novo deletions for a specific region of the Y chromosome. A candidate gene for the Y-chromosome azoospermia factor (AZF) has been identified and named Deleted in Azoospermia (DAZ). Here we describe the cloning and characterization of the Drosophila gene boule, which is a homologue of DAZ. The two genes encode closely related proteins that contain a predicted RNA-binding motif, and both loci are expressed exclusively in the testis. Loss of boule function results in azoospermia; meiotic divisions are blocked, although limited spermatid differentiation occurs. Histological examination of boule testes with cell-cycle markers indicates that the primary defect is at the meiotic G2/M transition. These results support the hypothesis that DAZ is the human AZF, and indicate that Boule and DAZ have an essential meiotic function in fly and human spermatogenesis.