Isolation of a cotton NADP(H) oxidase homologue induced by drought stress

The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L.) genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD) technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H) oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription.

Redes neurais artificiais para a modelagem do volume de madeira e biomassa do cerradão com dados de satélite

Resumo:O objetivo deste trabalho foi avaliar a eficácia da aplicação de modelos de análise de regressão e redes neurais artificiais (RNAs) na predição do volume de madeira e da biomassa acima do solo, da vegetação arbórea em área de cerradão. Volume de madeira e biomassa foram estimados com equações alométricas desenvolvidas para a área de estudo. Os índices de vegetação, como variáveis preditoras, foram estimados a partir de imagens do sensor LISS-III, e a área basal foi determinada por medições na floresta. A precisão das equações foi verificada pela correlação entre os valores estimados e observados (r), erro-padrão da estimativa (Syx) e gráfico residual. As equações de regressão para o volume de madeira total e do fuste (0,96 e 0,97 para r, e 11,92 e 9,72% para Syx, respectivamente) e para a biomassa (0,91 e 0,92 para r, e 22,73 e 16,80% para Syx, respectivamente) apresentaram bons ajustes. As redes neurais também apresentaram bom ajuste com o volume de madeira (0,99 e 0,99 para r, e 4,93 e 4,83% para Syx) e a biomassa (0,97 e 0,98 r, e 8,92 e 7,96% para Syx, respectivamente). A área basal e os índices de vegetação foram eficazes na estimativa do volume de madeira e biomassa para o cerradão. Os valores reais de volume de madeira e biomassa não diferiram estatisticamente dos valores estimados pelos modelos de regressão e redes neurais (χ2ns); contudo, as RNAs são mais acuradas.

Redes neurais artificiais para identificar genótipos de feijão-caupi semiprostrado com alta adaptabilidade e estabilidade fenotípicas

Resumo: O objetivo deste trabalho foi verificar a concordância entre as redes neurais artificiais (RNAs) e o método de Eberhart & Russel na identificação de genótipos de feijão-caupi (Vigna unguiculata) com alta adaptabilidade e estabilidade fenotípicas. Utilizou-se o delineamento experimental de blocos ao acaso com quatro repetições. Os tratamentos consistiram de 18 linhagens experimentais e duas cultivares de feijão-caupi. Foram conduzidos quatro ensaios de valor de cultivo e uso nos municípios de Aquidauana, Chapadão do Sul e Dourados, no estado do Mato Grosso do Sul. Os dados de produtividade de grãos foram submetidos às análises de variância individual e conjunta. Em seguida, os dados foram submetidos às análises de adaptabilidade e estabilidade por meio dos métodos de Eberhart & Russell e de RNAs. Houve elevada concordância entre os métodos avaliados quanto à discriminação da adaptabilidade fenotípica dos genótipos de feijão-caupi semiprostrado, o que indica que as RNAs podem ser utilizadas em programas de melhoramento genético. Em ambos os métodos avaliados, os genótipos BRS Xiquexique, TE97-304G-12 e MNC99-542F-5 são recomendados para ambientes desfavoráveis, gerais e favoráveis, respectivamente, por apresentarem produtividade de grãos acima da média geral dos ambientes e alta estabilidade fenotípica.

Isolado do vírus do mosaico do pepino obtido de bananeira no Estado de São Paulo pertence ao subgrupo Ia

Em 1996, foi feita a caracterização parcial de um isolado do vírus do mosaico do pepino (Cucumis mosaic virus, CMV) obtido de bananeira (Musa sp.) proveniente do município de Miracatu, SP. Com o objetivo de se determinar o subgrupo do isolado de CMV, recorreu-se às técnicas de ELISA, RT-PCR, RFLP e seqüenciamento de fragmentos de RNA genômico. Amostras de folhas infetadas, desidratadas com cloreto de cálcio e armazenadas à -20 °C desde 1994 na viroteca do Laboratório de Fitovirologia e Fisiopatologia, foram inoculadas em plantas de Nicotiana glutinosa. Dez dias após a inoculação, folhas apresentando mosaico foram utilizadas para DAS-ELISA e extração de RNAs totais. Em ELISA, houve reação apenas contra o anti-soro específico para CMV subgrupo I. Através de RT-PCR com primers desenhados para anelar em regiões conservadas da porção terminal 3' do gene da capa protéica, foi amplificado um fragmento de DNA com 486 pares de bases. O produto obtido via RT-PCR foi submetido à digestão com as enzimas EcoRI, HindIII, BamHI e MspI, obtendo-se um padrão de restrição esperado para o subgrupo I. Estes resultados foram confirmados através do seqüenciamento do produto de PCR, o qual apresentou homologia de 96% a 98% com os isolados do CMV pertencentes ao subgrupo I. Pelos sintomas observados na hospedeira diferencial Vigna unguiculata, o isolado foi confirmado como sendo do subgrupo Ia.

Effects of single and double infections with Potato virus X and Tobacco mosaic virus on disease development, plant growth, and virus accumulation in tomato

The tomato cv. Fukuju nº. 2 was used for studying the effect of single and double infections with Potato virus X (PVX) and Tobacco mosaic virus (TMV). Mixed infection resulted in a synergistic increase of disease severity, where more growth reduction was seen with simultaneous inoculations than with sequential inoculations at four-day intervals. At five and 12 days post-inoculation, the increased severity of the disease coincided with enhancement of virus accumulation in the rapidly expanding upper leaves. The PVX concentration in leaves nº 5 to 7 of doubly infected plants was three to six fold that of singly infected ones, as determined by DAS-ELISA. Mixed infection with the L strain led to higher enhancement of PVX than with the TMV-L11A strain. The concentration of TMV-L was lower in double infection and significantly higher than TMV-L11A in both singly and doubly infected plants. Analyses of the PVX ORF2 by Western blot and Northern hybridization revealed the pattern of accumulation of the 25 kDa protein and the RNAs, respectively, following those of the virion and coat protein. The strain TMV-L11A overcame the resistance gene in cv. GCR 237 (Tm-1). In the upper leaf nº. 8, the concentration of PVX was three times higher in plants with mixed infection than with L11A. The concentrations of the L and OM (TMV strains) in both singly and doubly infected plants were at very low levels, and the synergistic effect on PVX concentration and disease severity was not observed.

Viróides e virusóides: relíquias do mundo de RNA

Até meados do século XX, os vírus eram considerados os representantes mais simples da escala biológica. A descoberta dos RNAs satélites e dos viróides por volta de 1970 foi surpreendente, pois comprovou-se a existência de uma nova classe de moléculas auto-replicativas ainda mais simples, denominada agentes sub-virais. Há indícios de que os viróides e virusóides (que formam uma classe de RNAs satélites), teriam feito parte do "Mundo de RNA" (que precedeu o mundo atual baseado no DNA e proteínas), podendo ser considerados fósseis moleculares dessa era antiga. A simplicidade desses agentes sub-virais e o fato de que a molécula de RNA deve interagir diretamente com fatores do hospedeiro para o desenvolvimento do seu ciclo infeccioso colocam esses patógenos como um modelo para o estudo de processos metabólicos celulares. Nos últimos anos, tem-se observado um volume grande de publicações visando elucidar aspectos da interação viróide/hospedeiro, como os mecanismos da patogênese, movimento dos viróides nas plantas hospedeiras, silenciamento gênico e atividades das ribozimas. Mudanças recentes ocorridas na taxonomia desses patógenos com a criação de famílias, gêneros e espécies, além da descoberta de novos viróides, também têm sido verificadas. A presente revisão visa atualizar o leitor quanto aos recentes avanços nas pesquisas com viróides, principalmente na taxonomia, filogenia e em vários aspectos moleculares da interação viróide/hospedeiro. Estão incluídas também algumas características dos virusóides e sua relação evolutiva com os viróides.

Citrus exocortis viroid and Hop Stunt viroid Doubly infecting grapevines in Brazil

Viroids, non-protein-coding small (246-401 nt) circular single-stranded RNAs with autonomous replication, are currently classified into two families. Within the family Pospiviroidae, Citrus exocortis viroid (CEVd) belongs to the genus Pospiviroid while Hop stunt viroid (HSVd) is the single member of the genus Hostuviroid. These pathogens are distributed worldwide and infect a large number of hosts. In Brazil, isolates of CEVd and HSVd have been detected in both citrus and grapevine. To characterize and study the genetic variability of these viroids, total RNA from leaves of grapevine Vitis vinifera 'Cabernet Sauvignon' and V. labrusca 'Niagara Rosada' from Bento Gonçalves, RS, was used as a template for RT-PCR amplification with specific primers for the five viroids described infecting grapevines [HSVd, CEVd, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2) and Australian grapevine viroid (AGVd)]. Leaf samples of Citrus medica infected with CEVd from São Paulo were also analyzed. The resulting products were separated by agarose gel electrophoresis and DNA fragments of the expected size were eluted, cloned and sequenced. The grapevine samples analyzed were doubly infected by CEVd and HSVd. A phylogenetic analysis showed that the Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants, forming a specific group separated from citrus variants, whereas the Brazilian CEVd variants clustered with other citrus and grapevine variants.

Classification of antimicrobial resistance using artificial neural networks and the relationship of 38 genes associated with the virulence of Escherichia coli isolates from broilers

Avian pathogenic Escherichia coli (APEC) is responsible for various pathological processes in birds and is considered as one of the principal causes of morbidity and mortality, associated with economic losses to the poultry industry. The objective of this study was to demonstrate that it is possible to predict antimicrobial resistance of 256 samples (APEC) using 38 different genes responsible for virulence factors, through a computer program of artificial neural networks (ANNs). A second target was to find the relationship between (PI) pathogenicity index and resistance to 14 antibiotics by statistical analysis. The results showed that the RNAs were able to make the correct classification of the behavior of APEC samples with a range from 74.22 to 98.44%, and make it possible to predict antimicrobial resistance. The statistical analysis to assess the relationship between the pathogenic index (PI) and resistance against 14 antibiotics showed that these variables are independent, i.e. peaks in PI can happen without changing the antimicrobial resistance, or the opposite, changing the antimicrobial resistance without a change in PI.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

The RNA interference revolution

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia

MicroRNAs (miRNAs) are a class of small endogenous RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. miRNAs act in diverse biological processes including development, cell growth, apoptosis, and hematopoiesis, suggesting their association with cancer. We determined the miRNA expression profile of chronic and acute lymphocytic leukemias (CLL and ALL) using the TaqMan® MicroRNA Assays Human Panel (Applied Biosystems). Pooled leukemia samples were compared to pooled CD19+ samples from healthy individuals (calibrator) by the 2-DDCt method. Total RNA input was normalized based on the Ct values obtained for hsa-miR-30b. The five most highly expressed miRNAs were miR-128b, miR-204, miR-218, miR-331, and miR-181b-1 in ALL, and miR-331, miR-29a, miR-195, miR-34a, and miR-29c in CLL. To our knowledge, this is the first report associating miR-128b, miR-204 and miR-331 to hematological malignancies. The miR-17-92 cluster was also found to be up-regulated in ALL, as previously reported for some types of lymphomas. The differences observed in gene expression levels were validated for miR-331 and miR-128b in ALL and CD19+ samples. These miRNAs were up-regulated in ALL, in agreement with our initial results. A brief target analysis was performed for miR-331. One of its putative targets, SOCS1, promotes STAT activation, which is a known mediator of cell proliferation and survival, suggesting the possibility of an association between miR-331 and these processes. This initial screening provided information on miRNA differentially expressed in normal and malignant B-cells that could suggest the potential roles of these miRNAs in hematopoiesis and leukemogenesis.