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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Identificação de um fator do hospedeiro, RPS5A, envolvido na interação com a proteína de movimento (MP) de geminivírus / Identification of movement protein MP-intaracting host facotrs

Balmant, Kelly Mayrink 18 February 2011 (has links)
Made available in DSpace on 2015-03-26T13:42:20Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2368630 bytes, checksum: cd9eaf09470bcfbf6727027ecade3b46 (MD5) Previous issue date: 2011-02-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The movement protein MP from bipartite geminivirus (begomovirus) facilitates the cell-to-cell and long-distance transport of viral DNA in addition to affecting viral pathogenicity. To identify host factors that interact with MP, initially a cDNA library prepared from CaLCuV (Cabbage leaf curl virus)-infected Arabidopsis leaf mRNA was generated in a pEXPAD502 vector. To select for interactions between the bait BD-MP and cDNA library-encoded proteins, double transformants (BD-MP+ cDNA-AD) were plated on medium lacking histidine but supplemented with 3-aminotriazole (3-AT). From 5 x 105 transformants screened, two clones, encoding AtEXL3 (Exordium Like 3), a cell wall protein, and AtRPS5A, ribosomal protein S5A, displayed histidine/adenine auxotrophy and activate LacZ expression, on X-gal indicator plates. Expression of a full-length RPS5A cDNA fused to the Gal4 activation domain in yeast carrying BD-MP promoted growth of the double transformants in the absence of histidine and presence of 3-AT, in addition to activating high levels of LacZ expression. Furthermore, the interaction between MP and RPS5A was confirmed in vitro by pull down assays. Analysis of RPS5A gene expression by qRT-PCR demonstrated that the accumulation of rpS5A transcripts is suppressed by geminivirus infection. Based on these results and others, a functional model for the MP-RPS5A interaction is discussed. / A proteína de movimento (MP) de geminivírus bissegmentados (begomovirus) facilita o movimento célula-célula, bem como o movimento a longas distâncias do DNA viral, além de influenciar na patogenicidade viral. Com o objetivo de identificar fatores do hospedeiro que interagem com MP, inicialmente foi construída uma biblioteca de cDNA a partir de mRNAs de folhas de Arabidopsis infectadas com CaLCuV (Cabbage leaf curl virus) no vetor pEXPAD502. Para selecionar por interações entre a proteína quimérica BD-MP e proteínas codificadas pelos cDNAs da biblioteca, transformantes duplos (BDMP+ cDNA-AD) foram plaqueados em meio deficiente de histidina e suplementados com 3-amino triazol (3-AT). De um total de 5 x 105 transformantes escrutinados, dois clones, codificando EXL3 (Exordium Like 3), uma proteína de parede celular, e RPS5A, proteína ribossomal S5A, apresentaram auxotrofia a histidina e expressão do gene repórter LacZ, em placas indicadoras de X-gal. Expressão do cDNA completo de RPS5A fusionado ao domínio de ativação de Gal-4 em leveduras, carreando BD-MP, promoveu crescimento dos transformantes na ausência de histidina e presença de 3-AT, além de ativar altos níveis de expressão de LacZ.. Além disso, a interação entre MP e RPS5A foi confirmada in vitro por ensaios de pull down. Análises de expressão do gene RPS5A por meio de qRT-PCR demonstraram que o acúmulo de seus transcritos é reprimido por geminivírus. Baseado nestes resultados e de outros, um modelo funcional para interação de MP com RPS5A é discutido.

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