RESUMO
OBJECTIVE: Metformin, a medicine used for the treatment of type 2 diabetes, was previously reported to suppress age-dependent hyperproliferation of intestinal stem cells in Drosophila. Here, we aimed to investigate its anti-aging effects on other tissues, such as adult muscle and elucidate the mechanisms underlying the anti-ageing effect. MATERIALS AND METHODS: To evaluate the anti-muscle ageing effect of Metformin, we visualized ubiquitinated protein aggregates accumulated in adult muscle as the flies age by immunostaining and measured the total pixel size of the aggregates. We altered gene expression in the muscle by induction of dsRNA against the relevant mRNAs or mRNAs encoding the constitutively active mutant proteins using the Gal4/UAS system. We determined the mRNA levels by quantitative Real Time-Polymerase Chain Reaction (QRT-PCR). RESULTS: Continuous metformin feeding significantly extended the lifespan of Drosophila adults. Furthermore, the feeding suppressed the aging-dependent accumulation of ubiquitinated aggregates in adult muscle. To delineate the mechanism through which metformin influences the muscle aging phenotype, we induced the constitutively active AMPK specifically in the muscles and found that the activation of the AMPK-mediated pathway was sufficient for the anti-aging effect of Metformin. Furthermore, the AMPK-mediated downregulation of Tor-mediated pathways, subsequent induction of an eIF-4E inhibitor were involved in the effect. These genetic data suggested that the metformin effect is related to the partial suppression of protein synthesis in ribosomes. Furthermore, metformin stimulated autophagy induction in adult muscles. CONCLUSIONS: Our results suggest that metformin can be regarded as an anti-aging compound in Drosophila muscle. The stimulation of autophagy was also involved in the anti-aging effect, which delayed the progression of muscle aging in Drosophila adults.
Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Animais , Metformina/farmacologia , Drosophila/metabolismo , Adenilato Quinase , Proteínas Quinases Ativadas por AMP/metabolismo , EnvelhecimentoRESUMO
We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/citologia , Proteínas de Insetos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Alelos , Sequência de Aminoácidos , Animais , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Cromossomos/genética , Cromossomos/metabolismo , Sequência Conservada/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Genes de Insetos/genética , Guanosina Trifosfato/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/citologia , Larva/crescimento & desenvolvimento , Masculino , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Mapeamento Físico do Cromossomo , Poliploidia , RNA Mensageiro/análise , RNA Mensageiro/genética , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
OBJECTIVE: High-fat diet (HFD) feeding stimulates fat accumulation in mammals and Drosophila. In the present study, we examined whether simultaneous feeding of familiar anti-obesity drugs, quercetin glycosides (QG) and epigallocatechin gallate (EGCG), to Drosophila has the same suppressive effect on fat accumulation as previously reported in rats and mice. To understand the underlying molecular mechanisms of HFD diet-induced obesity and the suppression effect of the drugs, we performed transcriptome analyses. MATERIALS AND METHODS: We induced extra fat accumulation by feeding Drosophila fly food containing 20% coconut oil and quantified the triglyceride accumulated in flies. The effects of anti-obesity drugs were also evaluated. We isolated total RNA from each sample and performed RNA-seq analyses and quantitive Real Time-Polymerase Chain Reaction (qRT-PCR) to investigate altered gene expression. RESULTS: The mRNA levels of several genes involved in lipid metabolism, glycolysis/gluconeogenesis, and anti-oxidative stress changed in HFD-fed adults. Moreover, the levels altered in those fed an HFD with QG or EGCG. The qRT-PCR further confirmed the RNA-seq data, suggesting that the expression of five essential genes for lipid metabolism changed in HFD-fed flies and altered in the flies treated with anti-obesity drugs. The most remarkable alteration was observed in the dHSL gene encoding a lipase involved in lipid-storage after HFD feeding and HFD with QG or EGCG. These alterations are consistent with HFD-induced fat accumulation as well as the anti-obesity effects of the drugs in mammals, suggesting that the genes play an important role in anti-obesity effects. CONCLUSIONS: These are the first reports to date of entire profiles of altered gene expression under the conditions of diet-induced obesity and its suppression by anti-obesity drugs in Drosophila.
Assuntos
Fármacos Antiobesidade/administração & dosagem , Catequina/análogos & derivados , Metabolismo dos Lipídeos/efeitos dos fármacos , Obesidade/metabolismo , Quercetina/administração & dosagem , Animais , Peso Corporal/efeitos dos fármacos , Catequina/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Drosophila , Avaliação Pré-Clínica de Medicamentos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/administração & dosagem , Humanos , Masculino , Metabolômica/métodos , Obesidade/tratamento farmacológico , Obesidade/etiologia , Estresse Oxidativo/efeitos dos fármacos , Quercetina/análogos & derivados , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , Especificidade da EspécieRESUMO
OBJECTIVE: Sesamin is a major lignan constituent of sesame and possesses various health-promoting effects. Previous studies have demonstrated that sesamin extends the lifespan of Drosophila and Caenorhabditis elegans and corrects oxidative damage-related tissue dysfunction in mammals. To understand its anti-aging effects, we aimed to determine whether sesamin restores tissue function hampered by oxidative damage and suppresses several aging-related phenotypes using Drosophila senescence-accelerated models. MATERIALS AND METHODS: We elucidated the anti-aging effects of sesamin on several aging-related phenotypes in the muscle, brain and midgut using the senescence-accelerated models (Sod1n1 mutant and Sod1-depleted flies) by immunostaining experiments. We determined the expression levels of several anti-oxidative and DNA repair genes using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). We also identified the metabolite of sesamin in Drosophila by LC-MS/MS. RESULTS: We confirmed that sesamin (0.35 and 2 mg/ml) extended the lifespan of the fly models. As observed in mammals, it can be absorbed and metabolized by Drosophila adults. The sesamin feeding suppressed the age-dependent impairment of locomotor activity and inhibited the accumulation of reactive oxygen species (ROS) in their bodies. Sesamin delayed the age-dependent accumulation of damaged proteins in the muscle, partially suppressed the loss of dopaminergic neurons in adult brains displaying ROS accumulation, and suppressed the accumulation of DNA damage and hyperproliferation of intestinal stem cells. Four antioxidative genes and two DNA repair genes were simultaneously upregulated in sesamin-fed adults. CONCLUSIONS: These observations represent the first direct evidence of the anti-aging effects of sesamin at the individual level. We propose that sesamin exerts anti-aging effects in the muscles, brain and midgut by inducing antioxidative and DNA repair genes, resulting in extended lifespan in flies.
Assuntos
Envelhecimento/efeitos dos fármacos , Antioxidantes/farmacologia , Dioxóis/farmacologia , Modelos Animais de Doenças , Drosophila melanogaster , Intestinos , Lignanas/farmacologia , Longevidade , Envelhecimento/genética , Animais , Antioxidantes/análise , Antioxidantes/metabolismo , Células Cultivadas , Cromatografia Líquida , Dioxóis/análise , Dioxóis/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Intestinos/efeitos dos fármacos , Lignanas/análise , Lignanas/metabolismo , Músculos/efeitos dos fármacos , Músculos/metabolismo , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/metabolismo , Fenótipo , Superóxido Dismutase/deficiência , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Espectrometria de Massas em TandemRESUMO
The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.
Assuntos
Apoptose , Proteínas de Ligação a DNA/genética , DNA/biossíntese , Proteínas de Drosophila , Drosophila/genética , Proteínas de Insetos/genética , Células Fotorreceptoras de Invertebrados/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Bromodesoxiuridina/metabolismo , Divisão Celular , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Drosophila/fisiologia , Deleção de Genes , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas de Insetos/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Modelos Genéticos , Mutação , Fenótipo , Células Fotorreceptoras de Invertebrados/ultraestrutura , Complexo Repressor Polycomb 1 , Ligação Proteica , Fase SRESUMO
Transgenic flies in which ectopic expression of human p53 was targeted to the Drosophila eye imaginal disc were established. On sectioning of adult fly eyes which displayed a severe rough eye phenotype, most ommatidia were found to be fused and irregular shapes of rabdomeres were observed. In addition, many pigment cells were lost. In the developing eye imaginal disc, photoreceptor cell differentiation was initiated normally despite the ectopic expression of p53. However, expression of p53 inhibited cell cycle progression in eye imaginal disc cells and the S phase zone (the second mitotic wave) behind the morphogenetic furrow was almost completely abolished. Furthermore, expression of p53 induced extensive apoptosis of eye imaginal disc cells, and co-expression of baculovirus P35 in the eye imaginal disc suppressed the p53-induced rough eye phenotype. These results are consistent with the known functions of human p53 and indicate the existence of signaling systems with elements corresponding to human p53 in Drosophila eye imaginal disc cells. Genetic crosses of transgenic flies expressing p53 to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the p53-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify novel targets of p53 and its positive or negative regulators in Drosophila.
Assuntos
Olho/metabolismo , Fase S/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Sequência de Bases , Ciclo Celular/genética , Primers do DNA , Drosophila , Olho/crescimento & desenvolvimento , Olho/ultraestrutura , Humanos , Larva/crescimento & desenvolvimento , Microscopia Eletrônica de Varredura , Fenótipo , Deleção de SequênciaRESUMO
We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Doenças Neurodegenerativas/metabolismo , Peptídeos , Repetições de Trinucleotídeos/genética , Adenosina Trifosfatases , Animais , Apoptose/genética , Proteínas de Ciclo Celular/química , Modelos Animais de Doenças , Drosophila melanogaster/genética , Olho/crescimento & desenvolvimento , Olho/fisiopatologia , Mutação , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/genética , Fenótipo , Repetições de Trinucleotídeos/fisiologia , Proteína com ValosinaRESUMO
A spontaneous white mutation, white-milky (wmky) of Drosophila simulans is moderately unstable and is associated with a 16-kb long DNA insertion into the white gene. wmky, which is an unstable mutation found in D. simulans, has been genetically analyzed. Among nine spontaneous, partial reversions toward wild type, five were white locus mutations. They are phenotypically different from each other and three show eye color sexual dimorphism indicating a failure of the dosage compensation mechanism. Two w locus mutations whose eye color appeared identical between males and females were also isolated. Of the other back-mutants, three were associated with a recessive suppressor of wmky and one was a semidominant suppressor. These suppressor loci are located on the third chromosome at map positions about 90 and 120, respectively. The suppressor mutations demonstrate specific effects on w locus mutations derived from wmky which lack in the gene dosage compensation. Somatic instability was detected at the frequency of 5.6 X 10(-4) in wmky flies heterozygous for the recessive suppressor and the frequency was increased 10-fold when the suppressor mutation was placed in a different genetic background.
Assuntos
Elementos de DNA Transponíveis , Drosophila/genética , Pigmentação , Alelos , Animais , Mapeamento Cromossômico , DNA/genética , Olho , Mosaicismo , Mutação , Hibridização de Ácido Nucleico , Fenótipo , Supressão GenéticaRESUMO
Genetic and molecular characterization of the dominant suppressors of D-raf(C110) on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rl(Su23), was found to bear the same molecular lesion as rl(Sem), which has been reported to be dominant female sterile. However, rl(Su23) and the current stock of rl(Sem) showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Proteínas de Drosophila , Drosophila melanogaster/genética , MAP Quinases Reguladas por Sinal Extracelular , Proteínas Quinases Ativadas por Mitógeno/genética , Sequência de Aminoácidos , Animais , Cruzamentos Genéticos , Drosophila melanogaster/enzimologia , Olho/ultraestrutura , Feminino , Genes Dominantes , Heterozigoto , Homozigoto , Humanos , Infertilidade Feminina/genética , Masculino , Microscopia Eletrônica de Varredura , Proteínas Quinases Ativadas por Mitógeno/química , Dados de Sequência Molecular , Mutação , Proteínas Proto-Oncogênicas c-raf/genética , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Supressão GenéticaRESUMO
Eight alleles of Dsor1 encoding a Drosophila homologue of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppressed mutations for receptor tyrosine kinases (RTKs); torso (tor), sevenless (sev) and to a lesser extent Drosophila EGF receptor (DER). Furthermore, the Dsor1 alleles showed no significant interaction with gain-of-function mutations of DER. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrated that Dsor1 can activate yeast MAP kinase homologues if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes revealed that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Mutação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Sequência de Aminoácidos , Animais , Olho/enzimologia , Olho/ultraestrutura , Genes Dominantes , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Quinases/química , Homologia de Sequência de AminoácidosRESUMO
A temperature-sensitive (ts) mutation of Drosophila melanogaster for D-raf, encoding a serine/threonine protein kinase, was newly induced by EMS-treatment. Temperature-shift experiments on the ts mutant revealed that D-raf is required during most of the developmental stages, and confirmed the previously reported roles of D-raf in the regulation of cell proliferation and in the determination of cell fates at terminal regions of the embryo (Nishida et al., EMBO J. 7:775-781, 1988; Ambrosio et al., Nature 342:288-291, 1989a). Detailed analysis of cell proliferation demonstrated the role of D-raf at other than M-phase in cell cycle. TSP analysis during pupal stages revealed yet another role of D-raf in eclosion. Mosaic analysis of an eclosion-defective hypomorphic mutation revealed the tissue responsible for this defect to be the muscle and/or nervous system in the thorax. Molecular lesion associated with the ts mutation was found to be an alteration of an amino acid residue in a highly conserved region that defines the kinase subdomain VIII. Molecular analysis of null mutations also suggested the importance of the kinase domain for the biological functions of D-raf. Elucidation of the multi-functional nature of signal transducers is of great importance for our understanding of the molecular mechanisms of development, and the ts mutation for pleiotropic D-raf obtained in this study promises to be useful for dissecting signal transduction pathways during development.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Genes de Insetos , Proto-Oncogenes , Animais , Ciclo Celular , Divisão Celular , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário e Fetal/genética , Feminino , Masculino , Mutação , Transdução de Sinais , TemperaturaRESUMO
In phase-2 cells of diphasic Salmonella strains, expression of the phase-1 flagellin-encoding gene, fliC, is repressed by the repressor encoded by the fljA gene. Nine operator-constitutive (Oc) mutants of fliC were isolated from S. typhimurium by selecting those which could express fliC in the presence of the repressor. Among them, eight mutants could express fliC both in the presence and the absence of the repressor, whereas the ninth one could express only in the presence of the repressor. Nucleotide sequence analysis revealed that the Oc mutations of the former type were all located between bp 7 and 20 upstream from the coding region of fliC, which suggests that this region may correspond to the operator for fliC. The latter mutant was found to have a tandem duplication of 28 bp which contains a part of the operator sequence, and seems to require the repressor to activate fliC expression.
Assuntos
Proteínas de Bactérias/genética , Flagelina/genética , Genes Bacterianos , Mutação , Óperon , Salmonella typhimurium/genética , Sequência de Aminoácidos , Sequência de Bases , Genes Reguladores , Genótipo , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The transcription factor DREF regulates proliferation-related genes in Drosophila. With two-hybrid screening using DREF as a bait, we have obtained a clone encoding a protein homologous to human myelodysplasia/myeloid leukemia factor 1 (hMLF1). We termed the protein Drosophila MLF (dMLF); it consists of a polypeptide of 309 amino acid residues, whose sequence shares 23.1% identity with hMLF1. High conservation of 54.2% identity over 107 amino acids was found in the central region. The dMLF gene was mapped to 52D on the second chromosome by in situ hybridization. Interaction between dMLF and DREF in vitro could be confirmed by glutathione S-transferase pull-down assay, with the conserved central region appearing to play an important role in this. Northern blot hybridization analysis revealed dMLF mRNA levels to be high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae. This fluctuation of mRNA during Drosophila development is similar to that observed for DREF mRNA, except in the pupa and adult male. Using a specific antibody against the dMLF, we performed immunofluorescent staining of Drosophila Kc cells and showed a primarily cytoplasmic staining, whereas DREF localizes in the nucleus. However, dMLF protein contains a putative 14-3-3 binding motif involved in the subcellular localization of various regulatory molecules, and interaction with DREF could be regulated through this motif. The transgenic fly data suggesting the genetic interaction between DREF and dMLF support this possibility. Characterization of dMLF in the present study provides the molecular basis for analysis of its significance in Drosophila.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Southern Blotting , Proteínas de Ciclo Celular , Mapeamento Cromossômico , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Olho/metabolismo , Olho/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Humanos , Immunoblotting , Hibridização In Situ , Masculino , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Ligação Proteica , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
cDNAs encoding three Drosophila melanogaster MCM proteins, DmMCM3, DmMCM6 and DmMCM7, candidates of DNA replication-licensing factors, were cloned and sequenced. The deduced amino-acid sequences displayed 60, 59 and 68% identities with the respective Xenopus laevis homologues, XMCM3, XMCM6 and XMCM7. Six members of the D. melanogaster MCM family were found to share 31-36% identities in their amino-acid sequences, and to possess the five common domains carrying conserved amino-acid sequences as reported with X. laevis MCM proteins. DmMCM3, DmMCM6 and DmMCM7 genes were mapped to the 4F region on the X chromosome, the 6B region on the X chromosome and the 66E region on the third chromosome, respectively, by in situ hybridization. Contents of their mRNAs were proved to be high in unfertilized eggs and early embryos (0-4h after fertilization), then decrease gradually by the 12h time point, with only low levels detected at later stages of development except in adult females. This fluctuation pattern is similar to those of genes for proteins involved in DNA replication, such as DNA polymerase alpha and proliferating cell nuclear antigen, suggesting that expression of DmMCM genes is under the regulatory mechanism which regulates expression of other genes involved in DNA replication.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares , Cromossomo X , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/química , Mapeamento Cromossômico , Clonagem Molecular , Replicação do DNA , DNA Complementar , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/fisiologia , Larva , Componente 6 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xenopus laevis/genéticaRESUMO
A large body of data on molecular analyses of several multiallelic loci in Drosophila melanogaster has demonstrated a high incidence of mobile DNA element insertions among spontaneous mutations. In the sibling species D. simulans, the dispersed, middle repetitive, nomadic sequences are reduced to about one-seventh that of its sibling species (Dowsett and Young 1982). Does this reduced amount of middle repetitive DNA (or mobile DNA sequences) mean that in D. simulans the occurrence of insertion mutants will be rare compared with that of D. melanogaster? To test this possibility, we collected seven different spontaneous white mutants of D. simulans and studied their molecular gene structures. Five out of seven mutants had insertion sequences which varied in length from 0.4 kb to 16 kb. One bore a deletion spanning the w region and another showed no gross structural alteration. Thus the proportion of insertional mutations at the white locus in D. simulans is equivalent to that observed in D. melanogaster. Among the five insertional mutants, one, wmky, showed genetic instability; the other four were stable. wmky was found to mutate at a frequency of 2.1 x 10(-5) in meiotic cells and may also be unstable in somatic cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas do Olho/genética , Genes de Insetos/genética , Mutagênese Insercional/genética , Animais , Sequência de DNA Instável/genética , Drosophila melanogaster/genéticaRESUMO
We have found that mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. We have studied double mutant combinations between mutations affecting the activity of rolled/MAP kinase and several genes that are essential to the establishment of a bipolar spindle during progression through mitosis, and find no interactions with mutations in polo, mgr, or aurora. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors. We discuss these findings in relation to the proposed role of MAP kinase in mediating the spindle integrity checkpoint.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas de Drosophila , Drosophila/genética , MAP Quinases Reguladas por Sinal Extracelular , Genes de Insetos , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Drosophila/embriologia , Ativação Enzimática , Feminino , Fertilidade/genética , Masculino , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Mitose/fisiologia , Mutação , FenótipoRESUMO
Genetic and molecular investigations were carried out with 10 Japanese Drosophila melanogaster strains on P-M system of hybrid dysgenesis. The strains used here were collected in the years from 1952 to 1984 from various natural populations, and have been maintained in our laboratory. The whole genomic Southern hybridization was performed by using the 2.9-kb P element and the internal fragments as probes. Five strains possessed no P element copy and the other 5 strains possessed mainly incomplete P elements which had internal deletions. The former 5 strains were M, 2 of the latter were Q, and the remaining 3 were M' strains. Hikone-R, collected in 1952, had no P element copy, while Hikone-H, collected in 1957, was the earliest observed to possess multicopies of an incomplete P element. This revealed that P elements in Drosophila melanogaster were present more than 30 years ago in Japan, as already shown to have been the case on the American continent.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Variação Genética , Animais , Southern Blotting , Cruzamentos Genéticos , Feminino , Japão , Masculino , Reprodução/genética , Mapeamento por RestriçãoRESUMO
BACKGROUND: DNA replication-related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication- and proliferation-related genes in Drosophila. While the effects on the mutation in the DNA replication-related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild-type DREF activity in developing tissues have hitherto remained unclear. RESULTS: We over-expressed DREF in the wing imaginal disc using a GAL4-UAS targeted expression system in Drosophila. Over-expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF-induced notching wing phenotype. Furthermore, this was also the case with co-expression of baculovirus P35, a caspase inhibitor. In addition, over-expression of the 32 kDa boundary element-associated factor (BEAF-32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF-induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF-32 gene, exerted enhancing effects. To our knowledge, this is the first evidence for a genetic interaction between DREF and BEAF-32. CONCLUSION: The DREF-induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Proteínas do Olho , Fatores de Transcrição/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Apoptose/genética , Proteínas de Ligação a DNA/genética , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Larva , Neuropeptídeos/genética , Peptídeos/genética , Fenótipo , Fatores de Transcrição/metabolismo , Asas de Animais/anormalidades , Asas de Animais/patologiaRESUMO
Hybrids from the cross between males of Drosophila melanogaster and females of its sibling species (D. simulans, D. mauritiana, or D. sechellia) are embryonic lethal when they carry the wild type allele of zygotic hybrid rescue (zhr) from D. melanogaster. The zhr gene has been mapped in the proximal region of the X heterochromatin slightly distal to the proximal breakpoint of In(1)sc8, the region rich in 1.688 g/cm3 satellite DNA. Since this satellite DNA does not exist in the sibling species, the satellite DNA was considered to be involved in the hybrid lethality. We examined the hypothesis molecular cytogenetically. The results are (1) three Df(1)zhr chromosomes carried this satellite DNA, and (2) hybrids were viable even if the amount of the satellite DNA in hybrids was increased by adding minichromosomes Dp(1;f)1205 and Dp(1;f)1187 into the genome. These results do not support the above hypothesis.
Assuntos
Drosophila melanogaster/genética , Genes de Insetos , Hibridização Genética/genética , Alelos , Animais , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Cromossomos/química , Cromossomos/genética , Cruzamentos Genéticos , DNA Satélite/genética , Mecanismo Genético de Compensação de Dose , Feminino , Genes Letais , Genoma , Hibridização In Situ , Masculino , Dados de Sequência MolecularRESUMO
D-raf, a Drosophila homolog of Raf-1, plays key roles in multiple signal transduction pathways. Dsor1, a putative factor downstream of D-raf, was genetically identified by screening of dominant suppressors of D-raf. Dsor1Su1 mapped on X chromosome significantly suppressed the D-raf mutant phenotypes, and the loss-of-function mutations of Dsor1 showed phenotypes similar to those of the D-raf null mutations. Dsor1Su1 also significantly suppressed the mutations of other terminal class genes acting further upstream of D-raf. Molecular cloning of Dsor1 revealed its product with striking similarity to the microtubule-associated protein (MAP) kinase activator and yeast PBS2, STE7, and byr1. Our genetic results demonstrate the connection between raf and the highly conserved protein kinase cascade involving MAP kinase in vivo.