RESUMEN
The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis, the second largest parasitic killer in the world behind malaria. A large percentage of Leishmania membrane phospholipids is phosphatidylcholine (PC), formed via the Kennedy pathway, where the enzyme CTP: phosphocholine cytidylyltransferase (CCT) catalyzes the second, rate limiting step. Leishmania major CCT was expressed in non-pathogenic Leishmania tarentolae and exhibited activity that increased 10-fold in the presence of PC:oleate lipid vesicles. Confocal microscopy of L. tarentolae expressing L. major CCT fused to a red fluorescent protein revealed the enzyme is cytoplasmic but may associate with internal membranes. A truncated mutant of L. major CCT containing the catalytic domain was expressed in Escherichia coli and in vitro analysis of the enzyme showed catalysis was divalent cation-dependent and yielded a Vmax of 374 nmol/min/mg and Km values of 0.0648 mM and 3.74 mM, respectively, for the substrates CTP and phosphocholine.
RESUMEN
The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCE The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX's potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.
Asunto(s)
Antiinfecciosos , Rutenio , Aminoglicósidos/farmacología , Pseudomonas aeruginosa , Rutenio/farmacología , Plata/farmacología , Especies Reactivas de Oxígeno , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinfecciosos/farmacología , BacteriasRESUMEN
The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments into novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX®, a broad-spectrum silver containing antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX® could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX® on several antibiotic classes. We found that the combination of AGXX® and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX®/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX®/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX®/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials.
RESUMEN
Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif (RRM). Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully efficient MSUD response to unpaired genes. Additionally, at least one parent must have a functional sad-7 allele for a cross to produce ascospores. Although sad-7-null crosses are barren, sad-7Δ strains grow at a wild-type (wt) rate and appear normal under vegetative growth conditions. With respect to expression, sad-7 is transcribed at baseline levels in early vegetative cultures, at slightly higher levels in mating-competent cultures, and is at its highest level during mating. These findings suggest that SAD-7 is specific to mating-competent and sexual cultures. Although the role of SAD-7 in MSUD remains elusive, green fluorescent protein (GFP)-based tagging studies place SAD-7 within nuclei, perinuclear regions, and cytoplasmic foci of meiotic cells. This localization pattern is unique among known MSUD proteins and raises the possibility that SAD-7 coordinates nuclear, perinuclear, and cytoplasmic aspects of MSUD.
Asunto(s)
ADN de Hongos/genética , Proteínas Fúngicas/metabolismo , Silenciador del Gen , Meiosis/genética , Motivo de Reconocimiento de ARN , Alelos , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica , Genes Supresores , Proteínas Fluorescentes Verdes/metabolismo , Neurospora crassa/citología , Neurospora crassa/genética , Neurospora crassa/crecimiento & desarrollo , Esporas Fúngicas/genéticaRESUMEN
BACKGROUND: Hostile takeover (Hto) is a Drosophila protein trapping system that allows the investigator to both induce a gene and tag its product. The Hto transposon carries a GAL4-regulated promoter expressing an exon encoding a FLAG-mCherry tag. Upon expression, the Hto exon can splice to a downstream genomic exon, generating a fusion transcript and tagged protein. RESULTS: Using rough-eye phenotypic screens, Hto inserts were recovered at eight homeobox or Pax loci: cut, Drgx/CG34340, Pox neuro, araucan, shaven/D-Pax2, Zn finger homeodomain 2, Sex combs reduced (Scr), and the abdominal-A region. The collection yields diverse misexpression phenotypes. Ectopic Drgx was found to alter the cytoskeleton and cell adhesion in ovary follicle cells. Hto expression of cut, araucan, or shaven gives phenotypes similar to those of the corresponding UAS-cDNA constructs. The cut and Pox neuro phenotypes are suppressed by the corresponding RNAi constructs. The Scr and abdominal-A inserts do not make fusion proteins, but may act by chromatin- or RNA-based mechanisms. CONCLUSIONS: Hto can effectively express tagged homeodomain proteins from their endogenous loci; the Minos vector allows inserts to be obtained even in transposon cold-spots. Hto screens may recover homeobox genes at high rates because they are particularly sensitive to misexpression.
Asunto(s)
Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Animales , Bandeo Cromosómico , ADN Complementario/genética , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/metabolismo , Exones/genética , Ojo , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Femenino , Vectores Genéticos/genética , Proteínas de Homeodominio/biosíntesis , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Masculino , Modelos Genéticos , Fenotipo , Cromosomas Politénicos/ultraestructura , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Alas de Animales , Proteína Fluorescente RojaRESUMEN
Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination.
Asunto(s)
ADN de Hongos/genética , Proteínas Fúngicas/metabolismo , Recombinación Homóloga/genética , Neurospora crassa/genética , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cruzamientos Genéticos , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Homocigoto , Humanos , Meiosis , Mutagénesis Insercional , Neurospora crassa/citología , Neurospora crassa/crecimiento & desarrollo , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Esporas Fúngicas/crecimiento & desarrollo , Supresión GenéticaRESUMEN
The Drosophila melanogaster genome has been extensively characterized, but there remains a pressing need to associate gene products with phenotypes, subcellular localizations, and interaction partners. A multifunctional, Minos transposon-based protein trapping system called Hostile takeover (Hto) was developed to facilitate in vivo analyses of endogenous genes, including live imaging, purification of protein complexes, and mutagenesis. The Hto transposon features a UAS enhancer with a basal promoter, followed by an artificial exon 1 and a standard 5' splice site. Upon GAL4 induction, exon 1 can splice to the next exon downstream in the flanking genomic DNA, belonging to a random target gene. Exon 1 encodes a dual tag (FLAG epitope and mCherry red fluorescent protein), which becomes fused to the target protein. Hto was mobilized throughout the genome and then activated by eye-specific GAL4; an F1 screen for abnormal eye phenotypes was used to identify inserts that express disruptive fusion proteins. Approximately 1.7% of new inserts cause eye phenotypes. Of the first 23 verified target genes, 21 can be described as regulators of cell biology and development. Most are transcription factor genes, including AP-2, CG17181, cut, klu, mamo, Sox102F, and sv. Other target genes [l(1)G0232, nuf, pum, and Syt4] make cytoplasmic proteins, and these lines produce diverse fluorescence localization patterns. Hto permits the expression of stable carboxy-terminal subfragments of proteins, which are rarely tested in conventional genetic screens. Some of these may disrupt specific cell pathways, as exemplified by truncated forms of Mastermind and Nuf.
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Mapeo Cromosómico/métodos , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Transposasas/genética , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.
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Drosophila/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Animales , Drosophila/genética , Mutación , FenotipoRESUMEN
The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This "iab-8 RNA" produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only "in cis." Transcriptional interference with the abd-A promoter is the most likely mechanism.
Asunto(s)
Proteínas de Drosophila , MicroARNs/genética , Morfogénesis/genética , Proteínas Nucleares , ARN no Traducido/genética , Factores de Transcripción , Abdomen/crecimiento & desarrollo , Animales , Secuencia de Bases , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Infertilidad/genética , MicroARNs/metabolismo , Datos de Secuencia Molecular , Mutación , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt's B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V(H) and Sgamma3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci. CONCLUSIONS/SIGNIFICANCE: Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.
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Citidina Desaminasa/metabolismo , Histonas/metabolismo , Ubiquitinación/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Humanos , Ratones , Microscopía Confocal , Microscopía Fluorescente , Reacción en Cadena de la PolimerasaRESUMEN
Evidence for a developmental relationship between B cells and macrophages has led to the hypothesis that B cells evolved from a phagocytic predecessor. The recent identification of phagocytic IgM+ cells in fishes and amphibians supports this hypothesis, but raises the question of when, evolutionarily, was phagocytic capacity lost in B cells? To address this, leucocytes were isolated from red-eared sliders, Trachemys scripta, incubated with fluorescent beads and analysed using flow cytometry and confocal microscopy. Results indicate that red-eared slider B cells are able to ingest foreign particles and suggest that ectothermic vertebrates may use phagocytic B cells as part of a robust innate immune response.
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Linfocitos B/inmunología , Evolución Biológica , Fagocitos/inmunología , Tortugas/inmunología , Animales , Citometría de Flujo , Illinois , Microscopía ConfocalRESUMEN
Encephalitozoon species are the most common microsporidian pathogens of humans and domesticated animals. We recently discovered a new microsporidium, Encephalitozoon romaleae, infecting the eastern lubber grasshopper Romalea microptera. To understand its evolutionary relationships, we compared partial gene sequences of alpha- and beta-tubulin and methionine aminopeptidase 2 enzyme from this and related species. We also analyzed the rRNA internal transcribed spacer. Based on tubulin and MetAP-2 gene phylogenetic analysis, E. romaleae clustered with the Encephalitzoon group with strong bootstrap support (>99%). Within the Encephalitozoon clade, E. romaleae clustered with Encephalitozoon hellem for both the beta-tubulin and MetAP-2 phylogenies based on ML tree. The alpha-tubulin based ML tree, however, placed the new microsporidium closer to Encephalitozoon cuniculi. The rRNA internal transcribed spacer region of E. romaleae has 91% homology with E. hellem.
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Encephalitozoon/clasificación , Encephalitozoon/fisiología , Encefalitozoonosis/microbiología , Saltamontes/microbiología , Filogenia , Aminopeptidasas/genética , Animales , ADN de Hongos/genética , ADN Ribosómico/genética , ADN Espaciador Ribosómico/genética , Encephalitozoon/enzimología , Encephalitozoon/genética , Humanos , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , ARN Ribosómico/genética , Tubulina (Proteína)/genéticaRESUMEN
CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min (-1) mg (-1) and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 muM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg (2+) > Mn (2+) = Co (2+) > Ca (2+) = Ni (2+) > Zn (2+). The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.
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Núcleo Celular/enzimología , Citidililtransferasa de Colina-Fosfato/metabolismo , Proteínas de Drosophila/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/enzimología , Citidililtransferasa de Colina-Fosfato/química , Citidililtransferasa de Colina-Fosfato/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Dimethyl and diethyl carbaporphyrin ketals inhibit the growth of Leishmania tarentolae promastigotes in vitro. The concentration dependency of the inhibitory effect was tested using the MTT assay. The presence of reactive oxygen species, such as singlet oxygen and superoxide, was detected using electron paramagnetic resonance spectroscopy with selected spin traps and confocal microscopy in cultures exposed to these carbaporphyrin ketals. These unique porphyrinoids show promise as potent inhibitors of Leishmania.
Asunto(s)
Leishmania/efectos de los fármacos , Leishmaniasis/terapia , Fotoquimioterapia/métodos , Porfirinas/uso terapéutico , Animales , Espectroscopía de Resonancia por Spin del Electrón , Microscopía Confocal , Porfirinas/farmacología , Especies Reactivas de Oxígeno/análisis , TripanocidasRESUMEN
BACKGROUND: Within genus Drosophila, the endemic Hawaiian species offer some of the most dramatic examples of morphological and behavioral evolution. The advent of the Drosophila grimshawi genome sequence permits genes of interest to be readily cloned from any of the hundreds of species of Hawaiian Drosophila, offering a powerful comparative approach to defining molecular mechanisms of species evolution. A key step in this process is to survey the Hawaiian flies for characters whose variation can be associated with specific candidate genes. The wings provide an attractive target for such studies: Wings are essentially two dimensional, and genes controlling wing shape, vein specification, pigment production, and pigment pattern evolution have all been identified in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: We present a photographic database of over 180 mounted, adult wings from 73 species of Hawaiian Drosophila. The image collection, available at FlyBase.org, includes 53 of the 112 known species of "picture wing" Drosophila, and several species from each of the other major Hawaiian groups, including the modified mouthparts, modified tarsus, antopocerus, and haleakalae (fungus feeder) groups. Direct image comparisons show that major wing shape changes can occur even between closely related species, and that pigment pattern elements can vary independently of each other. Among the 30 species closest to grimshawi, diverse visual effects are achieved by altering a basic pattern of seven wing spots. Finally, we document major pattern variations within species, which appear to result from reduced diffusion of pigment precursors through the wing blade. CONCLUSIONS/SIGNIFICANCE: The database highlights the striking variation in size, shape, venation, and pigmentation in Hawaiian Drosophila, despite their generally low levels of DNA sequence divergence. In several independent lineages, highly complex patterns are derived from simple ones. These lineages offer a promising model system to study the evolution of complexity.
Asunto(s)
Drosophila/anatomía & histología , Alas de Animales/anatomía & histología , Animales , Femenino , Masculino , Especificidad de la EspecieRESUMEN
Methyl farnesoate (MF) appears to have important roles in the development, morphogenesis, and reproduction of crustaceans. To better understand the regulation of MF synthesis, we studied farnesoic acid O-methyltransferase (FAOMeT, the final enzyme in the MF biosynthetic pathway) in the American lobster (Homarus americanus). FAOMeT purified from mandibular organ (MO) homogenates had a MW of approximately 38,000. The sequences of trypsin fragments of purified FAOMeT were used to design PCR primers to amplify a cDNA fragment, which was used to isolate a full-length cDNA containing a single open reading frame (ORF) of 828 bp encoding a protein of 276 amino acids. The deduced amino acid sequence of this putative FAOMeT protein contained two copies of a conserved approximately 135 amino acid domain we term the CF (CPAMD8/FAOMeT) domain; single copies of this domain also occur in the human CPAMD8 protein (a member of the alpha-2 macroglobulin family) and an uncharacterized Drosophila protein. The recombinant protein had no FAOMeT activity. However, its addition to MO homogenates from eyestalk ablated (ESA) lobsters increased enzyme activity by up to 75%, suggesting that FAOMeT may require an additional factor or modification (e.g., phosphorylation) for its activation. The mRNA for the putative FAOMeT was primarily found in the proximal region of the MO, the predominant site of MF synthesis. FAOMeT transcripts were found in muscle tissue from ESA animals, but not in green gland, hepatopancreas, or in muscle tissue from intact animals. FAOMeT mRNA was also detected in embryos and larval stages. This is the first comprehensive report of this protein in the lobster, and is an important step in elucidating the functions of MF in these animals.