Deteção e caracterização molecular das estirpes de Wolbachia nos afídeos

A Wolbachiapertence a um grupo de bactérias intracelulares, transmitidas maternalmente, que se encontram amplamente distribuídas nos artrópodes. Estes endossimbiontes encontram-se normalmente nos tecidos do sistema reprodutor dos artrópodes e têm a capacidade manipular a sua reproduçãode modo a garantir a sua transmissão à descendência e rápida dispersão na população.A capacidade de manipulação reprodutiva da Wolbachia tornou-a o alvo de diversos estudos para uma maior e melhor perceção da sua implicação em processos biológicos e evolutivos e por acreditar-se que esta bactéria é uma promissora ferramenta no controlo de populações de insetos que são pragas agrícolas. Os afídeos são um grupo de insetos associados às plantas que podem ter um efeito devastador nas culturas agrícolas e hortícolas pois não só retiram nutrientes às plantas como podem ser vetores de doenças. Embora durante muito tempo se pensasse que estes insetos não albergavam a Wolbachia estudos recentes mostram que são várias as espécies de afídeos infetados com esta bactéria. O principal objetivo deste trabalho é estudar a prevalência de infeção por Wolbachia assim como a caracterização das suas estirpes em amostras de afídeos dos Arquipélagos da Madeira e dos Açores. Neste estudo foram analisadas 545 amostras de afídeos, 361 provenientes do Arquipélago da Madeira e 184 dos Açores. Utilizando a técnica da “Polymerase Chain Reaction” (reação em cadeia da polimerase) amplificou-se o gene 16S rRNA (RNA ribossomal) e verificou-se que 32 destas amostras encontravam-se infetadas com Wolbachia sendo a maior parte das amostras infetadas provenientes dos Açores. Para determinar a estirpe que infeta estes afídeos utilizou-se a tipagem sequencial multilocus (MLST) com os genesglutamil-tRNA amidotransferase, subunidade B (gatB), citocromo c oxidase, subunidade I (coxA), proteína hipotética conservativa (hcpA) e proteína da divisão celular (ftsZ). A análise filogenética realizada para os diferentes genes mostrou que grande parte das amostras analisadas estão incluídas em dois dos novos supergrupos descobertos para Wolbachia, supergrupo M e N.Foi detetada a presença da mesma estirpe de Wolbachia, supergrupo N, em duas espécies diferentes de afídeos, Neophyllaphis podocarpi e Aphis spiraecola da mesma planta hospedeira, Podocarpus macrophyllus. Esta infeção reforça a ideia de que a Wolbachia não recorre só a transmissão vertical para se difundir na população mas utiliza também a transmissão horizontal. A deteção e caracterização das estirpes de Wolbachia é essencial para um maior entendimento sobre a sua origem e forma de disseminação. Esta informação é importante para desenvolvimento de estratégias de controlo de pestes recorrendo a estes endossimbiontes.

Ecogenômica de archaea e monitoramento de comunidades de procariotos redutores de sulfato: aplicações na indústria de petróleo e gás

The human activities responsible for the ambient degradation in the modern world are diverse. The industrial activities are preponderant in the question of the impact consequences for brazilian ecosystems. Amongst the human activities, the petroliferous industry in operation in Potiguar Petroliferous Basin (PPB) displays the constant risk of ambient impacts in the integrant cities, not only for the human populations and the environment, but also it reaches the native microorganisms of Caatinga ground and in the mangrove sediment. Not hindering, the elaboration of strategies of bioremediation for impacted areas pass through the knowledge of microbiota and its relations with the environment. Moreover, in the microorganism groups associated to oil, are emphasized the sulfate-reducing prokaryotes (SRP) that, in its anaerobic metabolism, these organisms participate of the sulfate reduction, discharging H2S, causing ambient risks and causing the corrosion of surfaces, as pipelines and tanks, resulting in damages for the industry. Some ancestries of PRS integrate the Archaea domain, group of microorganisms whose sequenced genomes present predominance of extremophilic adaptations, including surrounding with oil presence. This work has two correlated objectives: i) the detection and monitoring of the gene dsrB, gift in sulfate-reducing prokaryotes, through DGGE analysis in samples of mDNA of a mangrove sediment and semiarid soil, both in the BPP; ii) to relate genomic characteristics to the ecological aspects of Archaea through in silico studies, standing out the importance to the oil and gas industry. The results of the first work suggest that the petrodegraders communities of SRP persist after the contamination with oil in mangrove sediment and in semiarid soil. Comparing the populations of both sites, it reveals that there are variations in the size and composition during one year of experiments. In the second work, functional and structural factors are the probable cause to the pressure in maintenance of the conservation of the sequences in the multiple copies of the 16S rDNA gene. Is verified also the discrepancy established between total content GC and content GC of the same gene. Such results relating ribosomal genes and the ambient factors are important for metagenomic evaluations using PCR-DGGE. The knowledge of microbiota associated to the oil can contribute for a better destination of resources by the petroliferous industry and the development of bioremediation strategies. Likewise, search to lead to the best agreement of the performance of native microbiota in biogeochemical cycles in Potiguar Petroliferous Basin ecosystem

Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae)

Leaf-cutting ants of the genera Atta and Acromyrmex (tribe Attini) are symbiotic with basidiomycete fungi of the genus Leucoagaricus (tribe Leucocoprineae), which they cultivate on vegetable matter inside their nests. We determined the variation of the 28S, 18S, and 5.8S ribosomal DNA (rDNA) gene loci and the rapidly evolving internal transcribed spacers 1 and 2 (ITS1 and ITS2) of 15 sympatric and allopatric fungi associated with colonies of 11 species of leafcutter ants living up to 2,600 km apart in Brazil. We found that the fungal rDNA and ITS sequences from different species of ants were identical (or nearly identical) to each other, whereas 10 GenBank Leucoagaricus species showed higher ITS variation. Our findings suggest that Atta and Acromyrmex leafcutters living in geographic sites that are very distant from each other cultivate a single fungal species made up of closely related lineages of Leucoagaricus gongylophorus. We discuss the strikingly high similarity in the ITS1 and ITS2 regions of the Atta and Acromyrmex symbiotic L. gongylophorus studied by us, in contrast to the lower similarity displayed by their non-symbiotic counterparts. We suggest that the similarity of our L. gongylophorus isolates is an indication of the recent association of the fungus with these ants, and propose that both the intense lateral transmission of fungal material within leafcutter nests and the selection of more adapted fungal strains are involved in the homogenization of the symbiotic fungal stock.

A search for markers of sugarcane evolution

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of a bisegmented double-stranded RNA virus (picobirnavirus) in calf faeces

To determine the incidence of rotavirus infection among dairy herds in the State of Sdo Paulo, Brazil, 576 faecal samples obtained from calves aged 1-45 days with and without diarrhoea, reared on 63 dairy cattle farms, were analyzed. Polyacrylamide gel electrophoresis (PAGE) identified 28 samples positive for group A rotavirus, while four samples, two diarrhoeic and two non-diarrhoeic, showed a bisegmented genome with a typical picobirnavirus pattern. Electron microscopy revealed spherical virus particles with a diameter of 37 nm and without a defined surface structure. The present study is the first report of a bisegmented virus identified in cattle in Brazil. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Caracterização de rizóbios indicados para produção de inoculantes por meio de sequenciamento parcial do 16S rRNA

O objetivo deste trabalho foi confrontar as sequências parciais do gene 16S rRNA de estirpes padrão de rizóbios com as de estirpes recomendadas para a produção de inoculantes no Brasil, com vistas à verificação da confiabilidade do sequenciamento parcial desse gene para a identificação rápida de estirpes. Foram realizados sequenciamentos através de reação em cadeia da polimerase (PCR) com iniciadores relativos à região codificadora do gene 16S rRNA entre as bactérias estudadas. Os resultados foram analisados pela consulta de similaridade de nucleotídeos aos do Basic Local Alignment Search Tool (Blastn) e por meio da interpretação de árvores filogenéticas geradas usando ferramentas de bioinformática. A classificação taxonômica das estirpes Semia recomendadas para inoculação de leguminosas com base em propriedades morfológicas e especificidade hospedeira não foi confirmada em todas as estirpes. A maioria das estirpes estudadas, consultadas no Blastn, é consistente com a classificação proposta pela construção de árvores filogenéticas das sequências destas estirpes, com base na similaridade pelo sequenciamento parcial do gene considerado.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2. 3, and 3short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform, 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain. (C) 2009 Elsevier B.V. All rights reserved.

An interaction between two RNA binding proteins, Nab2 and Pub1, links mRNA Processing/Export and mRNA stability

mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.

Molecular characterization of Blastocrithidia culicis L17 ribosomal protein

Blastocrithidia culicis is a protozoan of the family Trypanosomatidae. It is a parasite of insects, but the presence of bacteriumlike endosymbionts in its cytoplasm led some investigators to study this protozoan. This trypanosomatid does not infect humans and although it is phylogenetically distant from Trypanosoma cruzi, it presents many morphological characteristics, which are similar. In previous studies our group showed the presence of a L27 ribosomal protein in T cruzi (named TcrL27) using a RT-PCR, which also resulted in the cloning, sequencing and expression of an unexpected ribosomal protein, L17, in Blastocrithidia culicis (BcL17). In this paper, Western blot analysis demonstrated that the anti-BcL17 antibody recognizes the presence of the same ribosomal protein either in Blastochritidia culicis and T. cruzi nuclear extracts. Besides, two similar bands (40 and 47 kDa) appeared also in T. cruzi isolated ribosomal proteins and B. culicis nuclear extract corroborating with the findings showed in the phylogenetic reconstruction. With respect to their localization within the ribosome, both the L17 and L27 ribosomal proteins appear to belong to the peptidyl-transferase site, and are therefore part of the key step in protein synthesis. Both ribosomal proteins bind spiramycin derivatives, being therefore compounds of the macrolides connection sites in the ribosome. These findings would open a possibility to better evaluate this issue.

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

Identification of Staphylococcus saprophyticus isolated from patients with urinary tract infection using a simple set of biochemical tests correlating with 16S-23S interspace region molecular weight patterns

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interleukin 18 messenger RNA and proIL-18 protein expression in chorioamniotic membranes from pregnant women with preterm prelabor rupture of membranes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First identification of Sarcocystis tenella (Railliet, 1886) Moule, 1886 (Protozoa: Apicomplexa) by PCR in naturally infected sheep from Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do estresse térmico agudo sobre os níveis da proteína e RNA mensageiro da Hsp70, em fígado e cérebro de pintos de corte de diferentes linhagens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)