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2.
Arch Virol ; 152(5): 999-1007, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17238013

RESUMO

Lettuce mottle virus (LeMoV) and dandelion yellow mosaic virus (DaYMV) infect lettuce in South America and Europe, respectively. LeMoV and DaYMV possess isometric particles, occur at low concentrations in plants and have narrow host ranges. Partial genome sequences of both viruses were obtained using purified viral preparations and universal primers for members of the family Sequiviridae. DaYMV and LeMoV sequences were analyzed and showed identity with other members of the family. Universal primers that detect both viruses and specific primers for LeMoV and DaYMV were designed and used in RT-PCR-based diagnostic assays. These results provide the first molecular data on the LeMoV and DaYMV genomes and suggest that LeMoV is a member of the genus Sequivirus, probably distinct from DaYMV.


Assuntos
Genoma Viral , Doenças das Plantas/virologia , Sequivirus/classificação , Primers do DNA , Microscopia Eletrônica , Vírus do Mosaico/classificação , Vírus do Mosaico/genética , Sementes/virologia , Homologia de Sequência do Ácido Nucleico , Sequivirus/genética , Sequivirus/isolamento & purificação , Especificidade da Espécie
3.
J Virol Methods ; 79(2): 161-8, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10381086

RESUMO

A two-step reverse transcription reaction combined with long PCR was developed in order to obtain the full-length cDNA from the 12.2 kbp genomic RNA of rice tungro spherical virus. A first step reverse transcription, performed at 45 degrees C using a reverse transcriptase deprived of RNase H activity, allowed the synthesis of a nearly full-length cDNA of 11.7 kbp. A second step reaction, carried out at 65 degrees C using a thermostable polymerase, was necessary to destabilise secondary structures present at the 5' extremity of the RNA template which hampered the reverse transcription reaction in this region. The full-length cDNA obtained by the two-step reverse transcription was amplified successfully by long PCR and subsequently cloned into a plasmid vector. The cloned cDNA showed toxicity and proved to be unstable when amplified in E. coli.


Assuntos
DNA Viral/síntese química , Oryza/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequivirus/genética , Clonagem Molecular , DNA Complementar , Vetores Genéticos , Plasmídeos , RNA Viral
4.
Virology ; 247(1): 106-14, 1998 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-9683576

RESUMO

Rice tungro spherical virus encodes a large polyprotein containing motifs with sequence similarity to viral serine-like proteases and RNA polymerases. Polyclonal antisera raised against domains of the putative protease and polymerase in fusion with glutathione S-transferase detected a protein of about 35 kDa and, in very low amounts, a protein of about 70 kDa, respectively, in extracts from infected plants. In in vitro transcription/translation systems and in Escherichia coli we demonstrated a proteolytic activity in the C-terminal region of the polyprotein. This protease rapidly cleaved its polyprotein precursors in vitro. Mutating a potential cleavage site located N-terminal to the protease domain, Gln2526-Asp2527, diminished processing. The transversion mutation at the putative C-terminal cleavage site of the protease, at Gln2852-Ala2853, led to a delayed and partial processing.


Assuntos
Endopeptidases/metabolismo , Oryza/virologia , Sequivirus/genética , Sequivirus/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Endopeptidases/genética , Escherichia coli/genética , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genética
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