RESUMO
Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 µM), 52 for larvae (157 µM/3 µM) and more than 172 for females (>3.1 mM/18 µM). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration: 0.391 nM) was 46 000 times lower than the chronic LC50 for males; it was 115 000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Exposição Ambiental/análise , Imidazóis/toxicidade , Inseticidas/toxicidade , Nitrocompostos/toxicidade , Testes de Toxicidade , Animais , Feminino , Fertilidade/efeitos dos fármacos , Masculino , Modelos Animais , Neonicotinoides , Comportamento Sexual Animal/efeitos dos fármacos , Análise de Sobrevida , Testes de Toxicidade Aguda , Testes de Toxicidade CrônicaRESUMO
Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development. In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development. Here we show that the Dorsal switch protein 1 (DSP1), a Drosophila HMGB2 homologue, binds to a sequence present within Ab-Fab and in other characterized PREs. Addition of this motif to an artificial sequence containing Pleiohomeotic and GAGA factor consensus sites is sufficient for PcG protein recruitment in vivo. Mutations that abolish DSP1 binding to Ab-Fab and to a PRE from the engrailed locus lead to loss of PcG protein binding, loss of silencing, and switching of these PREs into constitutive TREs. The binding of DSP1 to PREs is therefore important for the recruitment of PcG proteins.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/classificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Animais , Sequência de Bases , Cromatina/genética , Imunoprecipitação da Cromatina , Cromossomos/genética , Cromossomos/metabolismo , Sequência Consenso/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas de Homeodomínio/genética , Hibridização in Situ Fluorescente , Mutação/genética , Complexo Repressor Polycomb 1 , Ligação Proteica , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Transgenes/genéticaRESUMO
The phosphatidylethanolamine binding proteins (PEBPs) family is evolutionarily conserved and involved in different physiological phenomena. PEBPs were found in many species from bacteria to mammals. Despite numerous studies, PEBPs' biological function and mode of action remain elusive. Based on sequence homology, seven PEBP genes were detected in the Drosophila genome. Only one of them, the odorant binding protein (OBP), has been characterized. To date nothing is known concerning the expression pattern and biological roles of the six other PEBP genes. By RT-PCR and Western blot analysis, we examined expression of the PEBPs in different tissues and embryos. The 6 PEBPs were differentially expressed. Only one, CG10298, is specific of only one tissue: the testis. Additionally, by comparing in wild type and male-sterile mutants we show that CG10298 is present only during spermatid differentiation. Furthermore, by comparing structural parameters of the six PEBP proteins with those of human PEBP-1, we have established that PEBP CG10298 is most closely related to human PEBP.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína de Ligação a Fosfatidiletanolamina/genética , Testículo/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/fisiologia , Masculino , Dados de Sequência Molecular , Proteína de Ligação a Fosfatidiletanolamina/classificação , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , RNA/análise , Homologia de Sequência , Espermátides/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Distribuição TecidualRESUMO
BACKGROUND: Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1. RESULTS: We show that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, we performed a genetic interaction analysis. We observed that corto mutants suppressed dsp11 sex comb phenotypes and enhanced AntpScx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, we found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells. CONCLUSION: Our results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 2 , Ligação Proteica/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoAssuntos
Drosophila melanogaster/química , Proteína de Ligação a Fosfatidiletanolamina/química , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Soluções , Homologia Estrutural de ProteínaRESUMO
DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Dicroísmo Circular , Clonagem Molecular , DNA/química , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Lisina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Triptofano/químicaRESUMO
The protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1: a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína HMGB1/genética , Proteínas de Grupo de Alta Mobilidade/genética , Animais , Cromatina/genética , Dicroísmo Circular , DNA/química , DNA/metabolismo , DNA Ligases/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Glutamina/química , Glutamina/genética , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Proteína HMGB2/química , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Plasmídeos , Ratos , Proteínas Recombinantes/genética , Transcrição GênicaRESUMO
Abnormal accumulation of alpha-synuclein filaments in Lewy bodies is a neuropathological hallmark of Parkinson's disease and sequestration of cellular protein into these protein aggregates may contribute to the degenerative process. We identified the transcriptional co-factor high mobility group protein 1 (HMGB-1) as a ligand for alpha-synuclein filaments by a filament spin-down technique, mass spectrometric peptide mapping and immunoblotting. HMGB-1 binds preferentially to aggregated alpha-synuclein and is present in alpha-synuclein filament-containing Lewy bodies isolated from brain tissue affected with dementia with Lewy bodies or Parkinson's disease. Our results demonstrate that alpha-synuclein filaments hold the potential for disturbing the cellular gene expression as they can sequester a component involved in cellular transcription regulation.
Assuntos
Proteína HMGB1/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteína HMGB1/análise , Proteína HMGB1/química , Proteína HMGB1/ultraestrutura , Humanos , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Isótopos de Iodo/metabolismo , Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/metabolismo , Ligantes , Microscopia Imunoeletrônica/métodos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/ultraestrutura , Neuritos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Mapeamento de Peptídeos/métodos , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Sinucleínas , alfa-SinucleínaRESUMO
In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10 10 80-µm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.
Assuntos
Drosophila melanogaster/anatomia & histologia , Imageamento por Ressonância Magnética/instrumentação , Microscopia/instrumentação , Imagem Corporal Total/instrumentação , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/instrumentação , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms: they control heterotrimeric G-proteins, inhibit the MAP-kinase and NFkappaB signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure-function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism: Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes' expression and the corresponding proteins' functions, and to studying physiological mechanisms such as development. Here, we describe an optimized protocol for high level over-expression and high yield/high purity production of CG18594, one of Drosophila six putative PEBPs, for biophysical studies. The yield of the purified 15N labeled protein is estimated to be 60 mg/L of M9 minimal medium. Analysis of the secondary structure using circular dichroism indicates that the protein comprises mainly beta-sheets at pH 7. The good dispersion of the crosspeaks on the 1H-15N HSQC spectrum provides evidence of a proper folding of the purified protein, though its time evolution suggests a tendency to denature. Taken together, these data are consistent with the assumption that the CG18594 protein belongs to the PEPB family.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína de Ligação a Fosfatidiletanolamina/genética , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Clonagem Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/isolamento & purificação , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
BACKGROUND INFORMATION: The Pc-G (Polycomb group) and trx-G (trithorax group) genes play a key role in the regulation of the homoeotic genes. The homoeotic gene Scr (Sex combs reduced) contained in the Antennapedia complex specifies segmental identity of the labial and prothoracic segments in Drosophila. Regulation of Scr requires the action of different enhancer elements spread over several kilobases. We previously identified an HMGB (high mobility group)-like protein DSP1 (dorsal switch protein 1), which works like a trx-G protein for the normal Scr expression. RESULTS: In the present study, we attempted to characterize the regulatory sequences involved in the maintenance of the Scr activation by DSP1. We report here, using a transgenic line for the Scr10.0XbaI-regulatory element, that lack of DSP1 affects the function of a reporter gene in legs' imaginal discs but not in embryos. We show by immunolocalization that DSP1 is recruited on polytene chromosomes to the insertion site of the transgene. Moreover, using chromatin immunoprecipitation experiments, we identify two regions of 1 kb in Scr10.0XbaI as the main DSP1 targets. CONCLUSION: These results provide strong evidence that the Scr gene expression is influenced by direct interaction between DSP1 and two Scr regulation elements. In addition, our results show that this interaction undergoes dynamic changes during development.