RESUMO
Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes the disease melioidosis. The disease can be fatal if left untreated or when antibiotic therapy is delayed and total clearance of the pathogen from the host is often not accomplished with current therapies. Thus, new therapeutic approaches for the treatment of infections caused by B. pseudomallei are required. To better understand host responses to B. pseudomallei infection, the activation of key proteins involved in the TLR inflammatory cascade was measured by western blotting. Activation of the mitogen-activated protein kinases (MAPKs) p38 and ERK were both significantly altered during both in vitro and in vivo infection. In considering an approach for therapy of B. pseudomallei infection the inhibition of ERK was achieved in vitro using the inhibitor PD0325901, along with decreased TNF-α production. However, the reduction in phosphorylated ERK and TNF-α release did not correspond with decreased bacterial replication or enhance clearance from infected macrophages. Despite this apparent lack of effect on the intracellular growth of B. pseudomallei in vitro, it is not clear what effect inhibition of ERK activation might have on outcome of disease in vivo. It may be that decreasing the levels of TNF-α in vivo could aid in reducing the overactive immune response that is known to ensue following B. pseudomallei infection, thereby increasing host survival.
Assuntos
Burkholderia pseudomallei/crescimento & desenvolvimento , Quimiocina CCL2/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Melioidose/patologia , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Benzamidas/farmacologia , Burkholderia pseudomallei/imunologia , Burkholderia pseudomallei/metabolismo , Linhagem Celular , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Macrófagos/microbiologia , Melioidose/imunologia , Melioidose/microbiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Francisella tularensis is a Gram-negative intracellular bacterium that causes the disease tularemia. The disease can be fatal if left untreated and there is currently no licenced vaccine available; the identification of new therapeutic targets is therefore required. Toll-like receptors represent an interesting target for therapeutic modulation due to their essential role in generating immune responses. In this study, we analysed the in vitro expression of the key mitogen-activated protein kinases (MAPKs) p38, JNK and ERK in murine alveolar macrophages during infection with F. tularensis. The phosphorylation profile of ERK highlighted its potential as a target for therapeutic modulation and subsequently the effect of ERK manipulation was measured in a lethal intranasal F. tularensis in vivo model of infection. The selective ERK1/2 inhibitor PD0325901 was administered orally to mice either pre- or post-challenge with F. tularensis strain LVS. Both treatment regimens selectively reduced ERK expression, but only the pre-exposure treatment produced decreased bacterial burden in the spleen and liver, which correlated with a significant reduction in the pro-inflammatory cytokines IFN-γ, MCP-1, IL-6, and TNF-α. However, no overall improvements in survival were observed for treated animals in this study. ERK may represent a useful therapeutic target where selective dampening of the immune response (to control the damaging pathology seen during infection) is combined with antibiotic treatment required to eradicate bacterial infection. This combination treatment strategy has been shown to be effective in other models of tularemia.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Interações Hospedeiro-Patógeno , Tularemia/patologia , Animais , Carga Bacteriana , Benzamidas/administração & dosagem , Linhagem Celular , Citocinas/metabolismo , Difenilamina/administração & dosagem , Difenilamina/análogos & derivados , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Fígado/microbiologia , Fígado/patologia , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/parasitologia , Camundongos Endogâmicos BALB C , Inibidores de Proteínas Quinases/administração & dosagem , Baço/microbiologia , Baço/patologia , Resultado do TratamentoRESUMO
Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring >8000 Acropora unigenes were used to follow gene expression changes during the 12h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common-an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.
Assuntos
Antozoários/crescimento & desenvolvimento , Antozoários/genética , Sequência de Aminoácidos , Animais , Antozoários/imunologia , Antozoários/fisiologia , Apoptose , Sequência de Bases , Cálcio/metabolismo , DNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/fisiologia , Lectinas/genética , Lectinas/imunologia , Metamorfose Biológica/genética , Metamorfose Biológica/fisiologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Estresse FisiológicoRESUMO
Mutations in the embryonic Drosophila Grapes/Chk1 checkpoint result in an abbreviated interphase, chromosome condensation defects and metaphase delays. To clarify the relationship between these phenotypes, we simultaneously timed multiple nuclear and cytoplasmic events in mutant grp-derived embryos. These studies support a model in which grp disrupts an S-phase checkpoint, which results in progression into metaphase with incompletely replicated chromosomes. We also show that chromosome condensation is independent of the state of DNA replication in the early embryo. Therefore, grp condensation defects are not a direct consequence of entering metaphase with incompletely replicated chromosomes. Rather, initiation of chromosome condensation (ICC) occurs at the normal time in grp-derived embryos, but the shortened interval between ICC and metaphase does not provide sufficient time to complete condensation. Our results suggest that these condensation defects, rather than incomplete DNA replication, are responsible for the extensive metaphase delays observed in grp-derived embryos. This analysis provides an example of how the loss of a checkpoint can disrupt the timing of multiple events not directly monitored by that checkpoint. These results are likely to apply to vertebrate cells and suggest new strategies for destroying checkpoint-compromised cancer cells.
Assuntos
Drosophila/genética , Membrana Nuclear/fisiologia , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Núcleo Celular/fisiologia , Quinase 1 do Ponto de Checagem , Segregação de Cromossomos , Replicação do DNA , Drosophila/embriologia , Proteínas de Drosophila , Genes de Insetos , Metáfase , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Fatores de TempoRESUMO
Immunoelectron microscopy with protein A gold has been used to determine the subcellular location of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. RESA was associated with dense vesicles presumed to be micronemes within merozoites. RESA was not detected on the surface of merozoites but was located at the membrane of erythrocytes infected with ring-stage parasites. RESA within merozoites was largely soluble in the nonionic detergent Triton X-100, but was insoluble in this detergent when associated with the erythrocyte membrane.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Malária/imunologia , Plasmodium falciparum/imunologia , Animais , Antígenos de Superfície/isolamento & purificação , Eritrócitos/imunologia , Eritrócitos/parasitologia , Eritrócitos/ultraestrutura , Humanos , Malária/sangue , Malária/parasitologia , Microscopia Eletrônica , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/ultraestruturaRESUMO
Members of the recently discovered ARID (AT-rich interaction domain) family of DNA-binding proteins are found in fungi and invertebrate and vertebrate metazoans. ARID-encoding genes are involved in a variety of biological processes including embryonic development, cell lineage gene regulation and cell cycle control. Although the specific roles of this domain and of ARID-containing proteins in transcriptional regulation are yet to be elucidated, they include both positive and negative transcriptional regulation and a likely involvement in the modification of chromatin structure.
Assuntos
Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Animais , Evolução Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , LevedurasRESUMO
The control of metazoan cell proliferation, a problem long the domain of cell culture studies, is now being examined in developing animals. Surprisingly, developmental regulation is mediated at a variety of cell-cycle stages. Highly conserved cell-cycle control mechanisms provide a focus for studying the regulatory processes involved.
Assuntos
Ciclo Celular/fisiologia , Animais , Ciclo Celular/genética , Drosophila melanogaster , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica/fisiologiaRESUMO
Recent advances in the preparation, control and measurement of atomic gases have led to new insights into the quantum world and unprecedented metrological sensitivities, e.g. in measuring gravitational forces and magnetic fields. The full potential of applying such capabilities to areas as diverse as biomedical imaging, non-invasive underground mapping, and GPS-free navigation can only be realised with the scalable production of efficient, robust and portable devices. We introduce additive manufacturing as a production technique of quantum device components with unrivalled design freedom and rapid prototyping. This provides a step change in efficiency, compactness and facilitates systems integration. As a demonstrator we present an ultrahigh vacuum compatible ultracold atom source dissipating less than ten milliwatts of electrical power during field generation to produce large samples of cold rubidium gases. This disruptive technology opens the door to drastically improved integrated structures, which will further reduce size and assembly complexity in scalable series manufacture of bespoke portable quantum devices.
RESUMO
Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFkappaB homolog Dorsal (DI). DI acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), it is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of DI by sequence-specific transcription factors such as Dead Ringer (Dri). We examined the interplay between DI, Gro and Dri on the hkb enhancer and show that when acting over a distance, Gro abolishes rather than converts DI activator function. Reducing the distance between DI- and Dri-binding sites, however, switches DI into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro - quenching and silencing of transcription - are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with DI. RTK signaling provides a way of modulating DI function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/fisiologia , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Proteínas Repressoras/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição , Animais , Animais Geneticamente Modificados , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Inativação Gênica , Mesoderma/metabolismo , Dados de Sequência Molecular , Morfogênese , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Transcrição GênicaRESUMO
We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others.
Assuntos
Sequência Conservada , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Drosophila , Proteínas de Homeodomínio/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Análise de SequênciaRESUMO
Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila melanogaster embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknüllt (zen) contains Dorsal binding sites as well as an essential element termed AT2. We have identified and purified an AT2 DNA binding activity in embryos and shown it to consist of cut (ct) and dead ringer (dri) gene products. Studies of loss-of-function mutations in ct and dri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/embriologia , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Proteínas Repressoras/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação/genética , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes de Insetos/genética , Proteínas de Homeodomínio/fisiologia , Proteínas de Insetos/metabolismo , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Oligodesoxirribonucleotídeos/metabolismo , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologiaRESUMO
The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames.
Assuntos
Antígenos de Protozoários/genética , Genes , Plasmodium falciparum/genética , Alelos , Sequência de Aminoácidos , Animais , Arrestina , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoRESUMO
Zygotic expression of the three rows (thr) gene of Drosophila melanogaster is required for normal cell proliferation during embryogenesis. Mitotic defects in thr mutant embryos begin during mitosis 15, and all subsequent divisions are disrupted. Chromosome disjunction and consequently cytokinesis fail during these defective mitoses, although the initial mitotic processes (chromosome condensation, spindle assembly, metaphase plate formation, and cyclin degradation) are not affected. Despite the failure of chromosome disjunction and cytokinesis, later mitotic events (chromosome decondensation) and subsequent cell cycle progression continue. The thr gene has been isolated and shown to encode a 1209 amino acid protein that shares no extended sequence similarity with known proteins. thr mRNA is present as maternal mRNA that degrades at the time of cellularization. At this and all subsequent times during embryogenesis, zygotic expression correlates with mitotic proliferation. These observations, together with the observation that the zygotic phenotype of thr mutant embryos is influenced by the maternal genotype, suggest that the embryonic phenotype results from exhaustion of the maternal thr contribution and does not reflect a developmentally restricted requirement for thr function. Our results indicate that the novel thr product is required specifically for chromosome disjunction during all mitoses.
Assuntos
Cromossomos/fisiologia , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Hormônios de Inseto/genética , Mitose/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular/fisiologia , Mapeamento Cromossômico , Clonagem Molecular , Ciclinas/análise , Drosophila melanogaster/embriologia , Expressão Gênica/fisiologia , Hibridização In Situ , Hormônios de Inseto/fisiologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , RNA Mensageiro/análise , RNA Mensageiro/genética , Fuso Acromático/química , Transformação Genética/genética , Tubulina (Proteína)/análiseRESUMO
We have generated and characterized a Drosophila cyclin E hypomorphic mutation, DmcycEJP, that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w+lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w+lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycEJP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog, cdc2c, retinoblastoma, or p21(CIP1) homolog dacapo, which encode proteins known to physically interact with Cyclin E, modified the DmcycEJP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhanced the DmcycEJP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhanced the DmcycEJP phenotype without affecting the number of cells entering S phase, but by decreasing the number of cells entering mitosis. Our analysis establishes the DmcycEJP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycEJP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development.
Assuntos
Ciclina E/genética , Ciclina E/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Mutação , Alelos , Animais , Ciclo Celular/genética , Divisão Celular/genética , Divisão Celular/fisiologia , Drosophila melanogaster/citologia , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Feminino , Genes de Insetos , Masculino , Microscopia Eletrônica de Varredura , Fenótipo , Fase S/genéticaRESUMO
pebble (pbl) is required for cytokinesis during postblastoderm mitoses (Hime, G., Saint, R., 1992. Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila. Development 114, 165-171; Lehner, C.F., 1992. The pebble gene is required for cytokinesis in Drosophila. J. Cell Sci. 103, 1021-1030) and encodes a putative guanine nucleotide exchange factor (RhoGEF) for Rho1 GTPase (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301-2314). Mutations in pbl result in the absence of a contractile ring leading to a failure of cytokinesis and formation of polyploid multinucleate cells. Analysis of the subcellular distribution of PBL demonstrated that during mitosis, PBL accumulates at the cleavage furrow at the anaphase to telophase transition when assembly of a contractile ring is initiated (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301-2314). In addition, levels of PBL protein cycle during each round of cell division with the highest levels of PBL found in telophase and interphase nuclei. Here, we report the expression pattern of pbl during embryonic development. We show that PEBBLE RNA and PBL protein have a similar tissue distribution and are expressed in a highly dynamic pattern throughout embryogenesis. We show that PBL is strongly enriched in dividing nuclei in syncytial embryos and in pole cells as well as in nuclei of dividing cells in postblastoderm embryos. Our expression data correlate well with the phenotypes observed in pole cells and, particularly, with the absence of cytokinesis after cellular blastoderm formation in pbl mutants.
Assuntos
Proteínas de Drosophila , Fatores de Troca do Nucleotídeo Guanina/genética , Animais , Drosophila/embriologia , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
In certain cases, homeobox genes with different in vivo roles encode proteins with similar in vitro DNA binding specificities. To test the role of the homeobox in the regulatory specificity of such genes, rough homeobox sequences were changed in part or entirely to those of the Deformed gene, and the modified rough genes tested for their ability to rescue the rough mutant phenotype. Surprisingly, the chimaeric genes retained levels of rough regulatory specificity but acquired no novel functions. These results suggest that factors other than the DNA binding specificity of the homeodomain play crucial roles in determining the target, and thus the regulatory specificity, of such proteins.
Assuntos
Drosophila melanogaster/genética , Olho/embriologia , Genes Homeobox , Genes Reguladores , Sequência de Aminoácidos , Animais , Sequência de Bases , Drosophila melanogaster/embriologia , Olho/anatomia & histologia , Dados de Sequência Molecular , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
Chromosomal INstability (CIN), a hallmark of cancer, refers to cells with an increased rate of gain or loss of whole chromosomes or chromosome parts. CIN is linked to the progression of tumors with poor clinical outcomes such as drug resistance. CIN can give tumors the diversity to resist therapy, but it comes at the cost of significant stress to tumor cells. To tolerate this, cancer cells must modify their energy use to provide adaptation against genetic changes as well as to promote their survival and growth. In this study, we have demonstrated that CIN induction causes sensitivity to metabolic stress. We show that mild metabolic disruption that does not affect normal cells, can lead to high levels of oxidative stress and subsequent cell death in CIN cells because they are already managing elevated stress levels. Altered metabolism is a differential characteristic of cancer cells, so our identification of key regulators that can exploit these changes to cause cell death may provide cancer-specific potential drug targets, especially for advanced cancers that exhibit CIN.
Assuntos
Instabilidade Cromossômica , Neoplasias/metabolismo , Estresse Fisiológico/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Dano ao DNA , Drosophila melanogaster , Embrião não Mamífero , Glutationa/metabolismo , Peroxidação de Lipídeos/genética , Neoplasias/genética , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Members of the Rh/T2/S-glycoprotein family of ribonuclease(RNase)-encoding genes have been found predominantly in fungi, plants and bacteria, where they have been implicated in functions as diverse as the phosphate-starvation response and self-incompatibility. We report the isolation and sequence of DmRNase-66B, the first member of this family to be found in an insect genome. This gene was identified by the analysis of a cDNA clone derived from cytological region 66B1-2 of the genome of Drosophila melanogaster. In a search of sequence databases for homologs of this gene, two animal viral proteins, gp53 of the bovine viral diarrhea virus (BVDV) and gp44/48 of the hog cholera virus (HCV), were also found to exhibit the characteristic features of this class of RNases. In all cases, the proteins contain two conserved pentameric amino-acid regions that have been shown to lie in the active site of these RNases. A series of Cys residues are also conserved in all members of this gene family. The discovery of members of this family of genes in an insect genome indicates that these RNases are widely conserved and play important roles in the animal, as well as the plant and prokaryotic kingdoms.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos/genética , Família Multigênica/genética , Ribonucleases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Endorribonucleases/genética , Genoma , Glicoproteínas/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Ribonucleases/classificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Vírus/genéticaRESUMO
We describe a new vector for the P-element-mediated introduction of gene constructs into the germ line of Drosophila melanogaster. The P-element vector carries 6.8 kb of genomic DNA containing the rough gene (ro) from D. melanogaster and a polylinker (MCS) containing ten unique cloning sites. To demonstrate its utility, we have cloned into the MCS of this vector, the firefly luciferase (Luc)-encoding gene (luc) under the control of the D. melanogaster hsp70 promoter and have transformed flies with the resultant P-element. Single insertions of this element, whether in the hemizygous or homozygous condition, completely rescued the ro- mutation and directed heat-inducible synthesis of Luc mRNA and enzyme.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Marcadores Genéticos , Vetores Genéticos , Proteínas Associadas aos Microtúbulos , Plasmídeos , Fatores de Transcrição/genética , Transformação Genética , Animais , Clonagem Molecular , Cruzamentos Genéticos , Feminino , Genes Dominantes , Masculino , Mapeamento por RestriçãoRESUMO
A cloned library of DNA complementary to the mRNA of adult Schistosoma japonicum has been prepared and expressed as fusion proteins with Escherichia coli beta-galactosidase. Colonies expressing the S. japonicum cDNA clones were screened both with antibodies from individuals with a history of schistosomiasis and with antibodies obtained from a rabbit immunized with whole adult worms. In both cases colonies were detected which bound antibody, although the frequency of antigen-positive clones was much higher with the rabbit antiserum than with human sera. In both cases the proportion of colonies reacting with antibodies was markedly lower than that published for equivalent screens of Plasmodium falciparum cDNA with sera from individuals with a history of falciparum malaria. Several major S. japonicum antigens were identified by the affinity purification of antibodies using immobilised fusion proteins produced during lytic growth of the recombinant bacteriophage.