RESUMO
AIMS: The importance of bacterioferritin in the virulence and pathogenicity of the genus Mycobacterium is still unclear. The aim of this study was to analyse if the expression of a recombinant bacterioferritin from M. tuberculosis (Mtb) by Mycma could improve the capacity of this bacillus to resist the host defence mechanisms. METHODS AND RESULTS: Recombinant Mycma, expressing bacterioferritin (Rv1876) from Mtb, was developed by transformation with pMIP12_Rv1876. To determine bacterioferritin influence on Mycma physiology and virulence, the mycobacteria growth was analysed in vitro and in vivo. It was observed that the expression of bacterioferritin improved the growth rate of recombinant Mycma_BfrA under iron excess and oxidative stress, as compared to the wild type. Furthermore, in the murine model of infection, it was observed that Mycma_BfrA-infected mice had higher bacillary load and a more pronounced lesion in the lungs when compared with the wild type. CONCLUSION: This study showed that bacterioferritin confers additional resistance to stress conditions, resulting in increased pathogenicity of Mycma during mice infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new insights about the importance of bacterioferritin in the virulence and pathogenicity of the Mycobacterium genus.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Ferritinas/metabolismo , Mycobacterium abscessus/fisiologia , Mycobacterium abscessus/patogenicidade , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Grupo dos Citocromos b/genética , Ferritinas/genética , Camundongos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium abscessus/genética , Mycobacterium abscessus/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , VirulênciaRESUMO
CuA is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, CuA is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima, σu* and πu, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states. A series of designed mutants span σu*/πu energy gaps ranging from 900 to 13 cm-1. The smallest gap corresponds to a variant with an effectively degenerate ground state. All engineered sites preserve the mixed-valence character of this metal center and the electron transfer functionality. An increase of the Cu-Cu distance less than 0.06 Å modifies the σu*/πu energy gap by almost 2 orders of magnitude, with longer distances eliciting a larger population of the πu state. This scenario offers a stark contrast to synthetic systems, as model compounds require a lengthening of 0.5 Å in the Cu-Cu distance to stabilize the πu state. These findings show that the tight control of the protein environment allows drastic perturbations in the electronic structure of CuA sites with minor geometric changes.
Assuntos
Proteínas de Bactérias/química , Complexos de Coordenação/química , Cobre/química , Grupo dos Citocromos b/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Grupo dos Citocromos b/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Elétrons , Estrutura Molecular , Engenharia de Proteínas , Subunidades Proteicas/química , Alinhamento de Sequência , Termodinâmica , Thermus thermophilus/enzimologiaRESUMO
In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.IMPORTANCEE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/genética , Ferritinas/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Reguladoras de Ferro/metabolismo , Ferro/metabolismo , RNA Bacteriano/metabolismo , Sinorhizobium meliloti/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/biossíntese , Grupo dos Citocromos b/biossíntese , Ferritinas/biossíntese , Proteínas Reguladoras de Ferro/genética , Mutação , RNA Bacteriano/genética , Sinorhizobium meliloti/genética , Fatores de Transcrição/genéticaRESUMO
Over the past decade, the Sea Catfish (Ariidae) genus Cathorops has been the focus of a major taxonomic review, which has resulted in the revalidation of five synonymized nominal species, and the recognition of seven new species. With 21 valid species, Cathorops is currently the most species-rich genus of Ariidae in the New World. The principal lacuna in the taxonomic knowledge of genus species is the uncertain status of Arius festae Boulenger, 1898, described from Naranjal, in the Guayas River basin of Ecuador. In the present study Cathorops festae is redescribed as a valid species based on morphological and molecular data.
Assuntos
Peixes-Gato/anatomia & histologia , Peixes-Gato/classificação , Distribuição Animal , Animais , Peixes-Gato/genética , Peixes-Gato/fisiologia , Grupo dos Citocromos b/genética , Equador , Feminino , Proteínas de Peixes/genética , Masculino , ATPases Mitocondriais Próton-Translocadoras/genética , Peru , Análise de Sequência de DNARESUMO
Avian Pathogenic Escherichia coli is responsible for significant economic losses in the poultry industry by causing a range of systemic or localized diseases collectively termed colibacillosis. The virulence mechanisms of these strains that are pathogenic in poultry and possibly pathogenic in humans have not yet been fully elucidated. This work was developed to study if over-expressed genes in a microarray assay could be potentially involved in the pathogenicity of an Avian Pathogenic Escherichia coli strain isolated from a swollen head syndrome case. For this study, five over-expressed genes were selected for the construction of null mutants [flgE (flagellar hook), tyrR (transcriptional regulator), potF (putrescine transporter), yehD (putative adhesin) and bfr (bacterioferritin)]. The constructed mutants were evaluated for their capacity for the adhesion and invasion of in vitro cultured cells, their motility capacity, and their pathogenic potential in one-day-old chickens compared with the wild-type strain (WT). The Δbfr strain showed a decreased adhesion capacity on avian fibroblasts compared with WT, in the presence and absence of alpha-D-mannopyranoside, and the ΔpotF strain showed decreased adhesion only in the absence of alpha-D-mannopyranoside. The ΔtyrR mutant had a reduced ability to invade Hep-2 cells. No mutant showed changes in invading CEC-32 cells. The mutants ΔflgE and ΔtyrR showed a decreased ability to survive in HD-11 cells. The motility of the mutant strains Δbfr, ΔyehD and ΔpotF was increased, while the ΔtyrR mutant showed reduction, and the ΔflgE became non-motile. No mutant strain caused the same mortality of the WT in one-day-old chickens, showing attenuation to different degrees.
Assuntos
Galinhas/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Doenças das Aves Domésticas/microbiologia , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Linhagem Celular , Embrião de Galinha , Grupo dos Citocromos b/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Feminino , Ferritinas/genética , Perfilação da Expressão Gênica/veterinária , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Regulação para Cima , Virulência , Fatores de Virulência/genéticaRESUMO
Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal bean (R + BP); Pontal bean + sweet potato (PB + SP); Pontal bean + pumpkin (PB + P); Pontal bean + rice + sweet potato (PB + R + P); Pontal bean + rice + sweet potato (PB + R + SP); positive control (Ferrous Sulfate). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE), gene expression of divalente metal transporter 1 (DMT-1), duodenal citocromo B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The test groups, except the PB, showed higher HRE (p < 0.05) than the control. Gene expression of DMT-1, DcytB and ferroportin increased (p < 0.05) in the groups fed with high content carotenoid crops (sweet potato or pumpkin). The PB group presented lower (p < 0.05) TAC than the other groups. The combination of rice and common beans, and those with high carotenoid content crops increased protein gene expression, increasing the iron bioavailability and antioxidant capacity.
Assuntos
Carotenoides/análise , Fabaceae/química , Alimentos Fortificados , Ferro/farmacocinética , Oryza/química , Animais , Disponibilidade Biológica , Carotenoides/administração & dosagem , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Regulação da Expressão Gênica , Hemoglobinas/metabolismo , História Antiga , Ferro/sangue , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fenóis/análise , Ácido Fítico/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transferrina/genética , Transferrina/metabolismoRESUMO
Prebiotics may increase intestinal Fe absorption in anaemic growing rats. The present study evaluated the effects of high-performance (HP) inulin and oligofructose on factors that regulate Fe absorption in anaemic rats during the growth phase. Male Wistar rats aged 21 d of age were fed AIN-93G ration without Fe for 2 weeks to induce Fe-deficiency anaemia. The rats were fed on day 35 a control diet, or a diet with 10 % HP inulin, or a diet with 10 % oligofructose, without Fe supplementation. The animals were euthanised after 2 weeks, and segments of the duodenum, caecum, colon and liver were removed. The expression levels of proteins in the intestinal segments were assessed using Western blotting. The levels of serum, urine and liver hepcidin and the concentrations of IL-10, IL-6 and TNF-α in the caecum, colon and liver were measured using the ELISA test. HP inulin increased the expression of the divalent metal transporter 1 protein in the caecum by 162 % (P= 0·04), and the expression of duodenal cytochrome b reductase in the colon by 136 % (P= 0·02). Oligofructose decreased the expression of the protein ferroportin in the duodenum (P= 0·02), the concentrations of IL-10 (P= 0·044), IL-6 (P= 0·036) and TNF-α (P= 0·004) in the caecum, as well as the level of urinary hepcidin (P< 0·001). These results indicate that prebiotics may interfere with the expression of various intestinal proteins and systemic factors involved in the regulation of intestinal Fe absorption in anaemic rats during the growth phase.
Assuntos
Anemia Ferropriva/dietoterapia , Proteínas de Transporte de Cátions/metabolismo , Grupo dos Citocromos b/metabolismo , Mucosa Intestinal/metabolismo , Prebióticos , Regulação para Cima , Anemia Ferropriva/imunologia , Anemia Ferropriva/metabolismo , Anemia Ferropriva/patologia , Animais , Proteínas de Transporte de Cátions/agonistas , Ceco/imunologia , Ceco/metabolismo , Ceco/patologia , Colo/enzimologia , Colo/imunologia , Colo/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Duodeno/imunologia , Duodeno/metabolismo , Duodeno/patologia , Hepcidinas/sangue , Hepcidinas/metabolismo , Hepcidinas/urina , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Inulina/efeitos adversos , Inulina/uso terapêutico , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Masculino , Oligossacarídeos/efeitos adversos , Oligossacarídeos/uso terapêutico , Tamanho do Órgão , Prebióticos/efeitos adversos , Ratos Wistar , Aumento de PesoRESUMO
Historical climate changes and orogenesis are two important factors that have shaped intraspecific biodiversity patterns worldwide. Although southern South America has experienced such complex events, there is a paucity of studies examining the effects on intraspecific diversification in this part of the world. Liolaemus pictus is the southernmost distributed lizard in the Chilean temperate forest, whose genetic structure has likely been influenced by Pleistocene glaciations. We conducted a phylogeographic study of L. pictus in Chile and Argentina based on one mitochondrial and two nuclear genes recovering two strongly divergent groups, Northern and Southern clades. The first group is distributed from the northernmost limit of the species to the Araucanía region while the second group is distributed throughout the Andes and the Chiloé archipelago in Southern Chile. Our results suggest that L. pictus originated 751 Kya, with divergence between the two clades occurring in the late Pleistocene. Demographic reconstructions for the Northern and Southern clades indicate a decrease in effective population sizes likely associated with Pleistocene glaciations. Surprisingly, patterns of genetic variation, clades age and historical gene flow in populations distributed within the limits of the Last Glacial Maximum (LGM) are not explained by recent colonization. We propose an "intra-Andean multiple refuge" hypothesis, along with the classical refuge hypothesis previously proposed for the biota of the Chilean Coastal range and Eastern Andean Cordillera. Our hypothesis is supported by niche modelling analysis suggesting the persistence of fragments of suitable habitat for the species within the limits of the LGM ice shield. This type of refuge hypothesis is proposed for the first time for an ectothermic species.
Assuntos
Mudança Climática , Evolução Molecular , Fagus , Lagartos/genética , Árvores , Animais , Chile , Grupo dos Citocromos b/genética , DNA Mitocondrial , Ecossistema , Haplótipos , Lagartos/classificação , Filogenia , FilogeografiaRESUMO
In Triatominae, "robustus" group constitutes a cluster of species with great haplotypic divergences but high similarities at morphological and nuclear DNA levels. Given these similarities, species identification generates a frequently problematic issue. In northwestern Amazonia, Rhodnius robustus cohabit with an apparently new species, cryptic with R. robustus (Abad-Franch and Monteiro, 2005). In this region (municipality of Puerto Asís, Department of Putumayo, Colombia), we collected insects classified as R. robustus by traditional keys. We compared this sample with specimens of R. robustus from Venezuela, and of R. prolixus from Colombia and Venezuela. The comparisons used landmark-based geometric morphometrics, and analyses of mitochondrial cytochrome b gene and of D2 variable region of the 28S RNA. The shape of the wings from Puerto Asís specimens disclosed clear-cut divergence from the shape of the wings as found for R. prolixus specimens from Venezuela and Colombia, and diverged from the shape of R. robustus from Venezuela. Thus, morphometric analyses suggested that the Puerto Asís collection could represent a new taxon. Using R. pallescens as an outgroup, a tentative phylogenetic tree based on the geometry of the wing showed the Rhodnius from Puerto Asís more similar to the R. prolixus from Colombia than their congeners from Venezuela. In contrast, the molecular classification clustered Colombian R. prolixus and Venezuelan R. robustus with published GenBank sequences, but it gave the insects from Puerto Asís a basal position to the "robustus" group. This outcome suggests that the Puerto Asís haplotype could be the one found by Abad-Franch and Monteiro (2005). Thus, both morphometric and molecular markers used here, although differing in the phylogenetic classification of samples, could differentiate the Puerto Asís sample from the morphologically similar R. prolixus and R. robustus. This could represent a valuable help in the entomological surveillance related to the control of Chagas disease in the South of Colombia and North of Ecuador.
Assuntos
Classificação/métodos , Grupo dos Citocromos b/genética , Insetos Vetores , RNA Ribossômico 28S/genética , Rhodnius , Animais , Doença de Chagas/transmissão , Colômbia , Equador , Feminino , Variação Genética , Insetos Vetores/anatomia & histologia , Insetos Vetores/classificação , Insetos Vetores/genética , Masculino , Filogenia , Rhodnius/anatomia & histologia , Rhodnius/classificação , Rhodnius/genética , Análise de Sequência de DNA , Especificidade da Espécie , Venezuela , Asas de Animais/anatomia & histologiaRESUMO
Trypanosoma cruzi, the etiologic agent for Chagas' disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.
Assuntos
Alquil e Aril Transferases/metabolismo , Grupo dos Citocromos b/metabolismo , Heme/análogos & derivados , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/genética , Trypanosoma cruzi/enzimologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Heme/biossíntese , Heme/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Reação em Cadeia da Polimerase , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Saccharomyces cerevisiae/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
The Arc (anoxic redox control) two-component signal transduction system, consisting of the ArcB sensor kinase and the ArcA response regulator, allows adaptive responses of Escherichia coli to changes of O(2) availability. The arcA gene was previously known as the dye gene because null mutants were growth sensitive to the photosensitizer redox dyes toluidine blue and methylene blue, a phenotype whose molecular basis still remains elusive. In this study we report that the toluidine blue O (TBO) effect on the arc mutants is light independent and observed only during aerobic growth conditions. Moreover, 16 suppressor mutants with restored growth were generated and analyzed. Thirteen of those possessed insertion elements upstream of the cydAB operon, rendering its expression ArcA independent. Also, it was found that, in contrast to cythocrome d, cythocrome o was not able to confer toluidine blue resistance to arc mutants, thereby representing an intriguing difference between the two terminal oxidases. Finally, a mechanism for TBO sensitivity and resistance is discussed.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos d/metabolismo , Citocromos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas Repressoras/genética , Cloreto de Tolônio/farmacologia , Anaerobiose , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Carotenoides/metabolismo , Catalase/metabolismo , Corantes/farmacologia , Grupo dos Citocromos b/genética , Grupo dos Citocromos d/genética , Citocromos/genética , Escuridão , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Glucose/farmacologia , Luz , Dados de Sequência Molecular , Mutação/genética , Oxirredutases/genética , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Superóxido Dismutase/metabolismoRESUMO
We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.
Assuntos
Quirópteros/parasitologia , DNA de Protozoário/genética , Genes de Protozoários/genética , Filogenia , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Tripanossomíase/veterinária , Animais , Brasil , Grupo dos Citocromos b/genética , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética , Genótipo , Histonas/genética , Cariotipagem , Camundongos , Dados de Sequência Molecular , Técnica de Amplificação ao Acaso de DNA Polimórfico , Triatominae/parasitologia , Trypanosoma cruzi/citologia , Tripanossomíase/parasitologiaRESUMO
The current taxonomic status of the species and subspecies belonging to the genus Alouatta is addressed by combined phylogenetic analysis using morphological, kariotipyc and molecular data (mitochondrial genes cytocrome oxidase II and cytochrome B). Our result demonstrated that Alouatta palliata is the most basal taxon for the genus in concordance with previous studies, as well as showing the validity of the taxon Alouatta sara as a species. Also our analysis shows that the sex chromosome has evolved from a XY/XX system to a X1X2Y1Y2/X1X1X2X2 system within the genus, as well as an increase in the size and complexity of the hioideal bone.
Assuntos
Animais , Alouatta/genética , Cromossomos Sexuais/genética , DNA Mitocondrial/genética , Filogenia , Alinhamento de Sequência , Alouatta/anatomia & histologia , Alouatta/classificação , Cariotipagem , Complexo IV da Cadeia de Transporte de Elétrons/genética , Evolução Molecular , Grupo dos Citocromos b/genéticaRESUMO
Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis--a simple, economical and rapid method--was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.
Assuntos
Cromossomos Humanos X/genética , Grupo dos Citocromos b/genética , Doença Granulomatosa Crônica/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Criança , Pré-Escolar , Doença Granulomatosa Crônica/genética , Humanos , Masculino , Mutação PuntualRESUMO
The current taxonomic status of the species and subspecies belonging to the genus Alouatta is addressed by combined phylogenetic analysis using morphological, kariotipyc and molecular data (mitochondrial genes cytocrome oxidase II and cytochrome B). Our result demonstrated that Alouatta palliata is the most basal taxon for the genus in concordance with previous studies, as well as showing the validity of the taxon Alouatta sara as a species. Also our analysis shows that the sex chromosome has evolved from a XY/XX system to a X1X2Y1Y2/X1X1X2X2 system within the genus, as well as an increase in the size and complexity of the hioideal bone.
Assuntos
Alouatta/genética , DNA Mitocondrial/genética , Filogenia , Cromossomos Sexuais/genética , Alouatta/anatomia & histologia , Alouatta/classificação , Animais , Grupo dos Citocromos b/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Evolução Molecular , Cariotipagem , Alinhamento de SequênciaRESUMO
The photosynthetic protist Euglena gracilis contains chloroplasts surrounded by three membranes which arise from secondary endosymbiosis. The genes petA and petD, encoding cytochrome f and subunit IV of the cytochrome bf complex, normally present in chloroplast genomes, are lacking from the chloroplast DNA (cpDNA) of E. gracilis. The bf complex of E. gracilis was isolated, and the identities of cytochrome f and subunit IV were established immunochemically, by heme-specific staining, and by Edman degradation. Based on N-terminal and conserved internal protein sequences, primers were designed and used for PCR gene amplification and cDNA sequencing. The complete sequence of the petA cDNA and the partial sequence of the petD cDNA from E. gracilis are described. Evidence is provided that in this protist, the petA and petD genes have migrated from the chloroplast to the nucleus. Both genes exhibit a typical nuclear codon usage, clearly distinct from the usage of chloroplast genes. The petA gene encodes an atypical cytochrome f, with a unique insertion of 62 residues not present in other f-type cytochromes. The petA gene also acquired a region that encodes a large tripartite chloroplast transit peptide (CTP), which is thought to allow the import of apocytochrome f through the three-membrane envelope of E. gracilis chloroplasts. This is the first description of petA and petD genes that are nucleus-localized.
Assuntos
Núcleo Celular/metabolismo , Cloroplastos/genética , Grupo dos Citocromos b/genética , Complexo Citocromos b6f , Citocromos/genética , Euglena gracilis/genética , Genoma de Planta , Fotossíntese , Sequência de Aminoácidos , Animais , Citocromos f , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Subunidades ProteicasRESUMO
The phylogeographical structure of the closely related species Rhodnius prolixus and R. robustus is presented based on a 663-base pair (bp) fragment of the mitochondrial cytochrome b gene. Twenty haplotypes were recovered from 84 samples examined, representing 26 populations from seven Latin American countries. The resulting phylogenetic tree is composed of five major reciprocally monophyletic clades, one representing R. prolixus and four representing R. robustus. While R. prolixus is a very homogeneous assemblage, R. robustus has deeper clades and is paraphyletic, with the clade comprising R. robustus from Venezuela (Orinoco region) more closely related to the R. prolixus clade than to the other R. robustus populations from the Amazon region. The R. robustus paraphyly was supported further by the analysis of a nuclear gene (D2 region of the 28S RNA) for a subset of specimens. The data support the view that R. robustus represents a species complex. Levels of sequence divergence between clades within each region are compatible with a Pleistocene origin. Nucleotide diversity (pi) for all R. prolixus populations was extremely low (0.0008), suggesting that this species went through a recent bottleneck, and was subsequently dispersed by man.
Assuntos
Filogenia , Rhodnius/genética , Rhodnius/fisiologia , Animais , Sequência de Bases , Grupo dos Citocromos b/genética , Primers do DNA , Geografia , Dados de Sequência Molecular , RNA Ribossômico 28S/genética , Rhodnius/classificação , América do SulRESUMO
A study of genetic diversity at microsatellite loci and the mitochondrial DNA (mtDNA) cytochrome b gene was carried out to assess genetic relationships among four Mexican cave (Pachon, Sabinos, Tinaja, Chica) and four surface populations of Astyanax fasciatus (Characidae) from northeast Mexico and the Yucatan. With the exception of Chica, the cave populations were all characterized by extremely low microsatellite variability, which most likely resulted from bottleneck events. Population analyses of the microsatellite data indicated no measurable levels of gene flow between all cave and surface populations (F(ST) > 0.0707). Phylogenetic analyses of mtDNA data showed that only two cave populations - Sabinos and Tinaja - group together to the exclusion of surface populations. From the microsatellite data these cave populations cluster with the Pachon cave fish population. The mtDNA thus appears to have been replaced in Pachon because of introgressive hybridization. It is likely that these three cave populations have descended from a surface ancestor in common with current surface populations, rather than evolving recently from one of the extant surface populations. Like Pachon, the Chica population clustered with the surface populations according to mtDNA data, but was not clearly associated with either the surface or the other cave populations according to the microsatellite data. Our data indicate that the Chica population evolved recently from a surface population, and subsequently hybridized with a phylogenetically older cave population. In conclusion, both the microsatellite and mtDNA data suggest multiple origins of cave populations and the Chica and Sabinos/Tinaja/Pachon were founded after at least two independent invasions from surface populations.
Assuntos
DNA Mitocondrial/genética , Peixes/genética , Genética Populacional , Repetições de Microssatélites/genética , Animais , Sequência de Bases , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , DNA Mitocondrial/química , Evolução Molecular , Variação Genética , México , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The spiny rat Mesomys hispidus is one of many South American rodents that lack adequate taxonomic definition. The few sampled populations of this broadly distributed trans-Amazonian arboreal rat have come from widely separated regions and are typically highly divergent. The holotype was described in 1817 by A.-G. Desmarest, after Napoleon's army brought it to Paris following the plunder of Lisbon in 1808; however, the locality of origin has remained unknown. Here we examine the taxonomic status of this species by direct comparison of 50 extant individuals with the holotype at the morphometric and genetic levels, the latter based on 331 bp of the mitochondrial cytochrome b gene retrieved from a small skin fragment of the holotype with ancient DNA technology. Extensive sequence divergence is present among samples of M. hispidus collected from throughout its range, from French Guiana across Amazonia to Bolivia and Peru, with at least seven mitochondrial clades recognized (average divergence of 7.7% Kimura 2-parameter distance). Sequence from the holotype is, however, only weakly divergent from those of recent samples from French Guiana. Moreover, the holotype clusters with greater that 99% posterior probability with samples from this part of Amazonia in a discriminant analysis based on 22 cranial and dental measurements. Thus, we suggest that the holotype was originally obtained in eastern Amazonia north of the Amazon River, most likely in the Brazilian state of Amapá. Despite the high level of sequence diversity and marked morphological differences in size across the range of M. hispidus, we continue to regard this assemblage as a single species until additional samples and analyses suggest otherwise.
Assuntos
Filogenia , Roedores/classificação , Animais , Sequência de Bases , Biometria , Análise por Conglomerados , Grupo dos Citocromos b/genética , Geografia , Funções Verossimilhança , Dados de Sequência Molecular , Roedores/anatomia & histologia , Roedores/genética , América do SulRESUMO
The pelagic larvae of many marine organisms can potentially disperse across hundreds of kilometers, but whether oceanographic or behavioral mechanisms can constrain dispersal over periods sufficient for the evolution of genetic differentiation remains unclear. Here, we concurrently examine larval duration and genetic population differentiation in a cleaner goby, Elacatinus evelynae, a member of the most species-rich genus of Caribbean reef fishes. Despite evidence for extended pelagic duration (21 days), populations of E. evelynae show strong genetic differentiation: among color forms (1.36 to 3.04% divergent at mitochondrial cytochrome b) and among island populations within color forms (Phi(ST) up to 70%). These results suggest that marine populations can remain demographically closed for thousands of generations despite extended larval duration, and that recognition cues such as color may promote speciation when geographic barriers are transient or weak.