A polymerase chain reaction method adapted for selective amplification and cloning of 3' sequences of potyviral genomes: application to dasheen mosaic virus

Pappu, S. S.; Brand, Reon J.; Pappu, H. R.; Rybicki, Edward P.; Gough, K. H.; Frenkel, M. J.; Niblett, C.L.

doi:10.1016/0166-0934(93)90158-n

Cited by 133 publications

(78 citation statements)

References 39 publications

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“…Reverse transcription (RT) and PCR amplification (Saiki et al, 1985) reactions (RT PCR) were performed as previously described (Pappu et al, 1993b). The amplified fragment was cloned into SmaIcut pUCll8 and sequenced as previously described…”

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Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus

Pappu

Rybicki

et al. 1994

Journal of General Virology

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The T-terminal region of a Florida isolate of dasheen mosaic potyvirus (DMV-LA) genome including the coat protein (CP) gene was cloned and sequenced. Protease digestion was predicted to occur between the glutamine and alanine residues at positions 79 and 80 of the 408 residue long polypeptide to produce a CP of 329 amino acids with an estimated 3//,. of 36229. Following the putative protease recognition site is a DAG sequence, which is conserved among aphid-transmitted potyviral CPs. There is an unusual and unique stretch of 52 amino acids after the DAG that is repetitive and rich in threonine and asparagine. A sequence of 10 residues (GNNTNTNT2T) was repeated three times in tandem within this stretch and was followed by six proline residues. Several potential glycosylation sites were found clustered within this region. Expression in Escherichia co//and Western blotting of the CP confirmed its size and serological identity. Sequence comparisons and phylogenetic reconstructions indicated that DMV is a distinct potyvirus within the passionfruit woodiness virus subgroup cluster.

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“…Crude viral nucleic acids were prepared from leaf tissue infected with the Florida DMV isolate LA (Pappu et al, 1993b). Reverse transcription (RT) and PCR amplification (Saiki et al, 1985) reactions (RT PCR) were performed as previously described (Pappu et al, 1993b).…”

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Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus

Pappu

Rybicki

et al. 1994

Journal of General Virology

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“…A mistura foi aquecida a 70 ºC por 10 min, resfriada à temperatura ambiente e aplicada à coluna do "RNeasy Plant Mini Kit" conforme especificação do fabricante (Qiagen). Para a síntese de DNA complementar (cDNA) foram utilizados os oligonucleotídeos CN47, CN54 e CN55, contendo 21 timinas antecedidas, respectivamente, por A, C ou G (Pappu et al, 1993) complementares à cauda poliadenilada da região 3' terminal do RNA viral. Na reação (20 ?…”

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Caracterização do gene da proteína capsidial do Grapevine virus A em videiras afetadas pela acanaladura do lenho de Kober no Estado de São Paulo

Moreira

Gaspar

Camargo³

et al. 2004

Fitopatol. bras.

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O presente trabalho caracteriza o gene codificador da proteína capsidial do isolado do Grapevine virus A (GVA) encontrado no Estado de São Paulo (GVA-SP). RNA total foi extraído de folhas e pecíolos de plantas de videira (Vitis spp.) da variedade 'Kober 5BB' e submetido a RT-PCR usando oligonucleotídeos desenhados para amplificar um fragmento entre as posições 6409 e 7175 do RNA do GVA ("GenBank", acesso X75433). Foi obtido um fragmento de tamanho esperado (767 nt) que inclui o gene da proteína capsidial, codificando 198 aminoácidos. A seqüência do GVA-SP apresentou similaridade de nucleotídeos e aminoácidos de, respectivamente, 86-92,3% e 94,5-98% com isolados do GVA da Europa, África e Japão (Acessos X75433, AF441234, AF007415, AB039841) e da região Sul do Brasil (Acesso AF494187), sendo, entretanto, mais similar aos isolados africano e italiano.

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“…Electron microscopy is also useful [2], but is normally suited for morphological diagnosis such as distinguishing a rod-shaped virus from an icosahedron. Serological, hybridization, and PCR techniques are easy methods available to identify viruses but require advanced knowledge of capsid protein antigenicity or nucleic acid sequence, or require availability of a range of antisera needed to characterize one of many possible viruses [3][4][5]. Direct sequencing of the virus can also be used and may be the most accurate diagnostic tool, although this requires the initial work of cloning, subcloning, or primer walking.…”

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Investigative proteomics: Identification of an unknown plant virus from infected plants using mass spectrometry

Cooper

Eckert

Andon

et al. 2003

J. Am. Soc. Mass Spectrom.

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We describe the identification of a previously uncharacterized plant virus that is capable of infecting Nicotiana spp. and Arabidopsis thaliana. Protein extracts were first prepared from leaf tissue of uninfected tobacco plants, and the proteins were visualized with two-dimensional electrophoresis (2-DE). Matching gels were then run using protein extracts of a tobacco plant infected with tobacco mosaic virus (TMV). After visual comparison, the proteins spots that were differentially expressed in infected plant tissues were cut from the gels and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Tandem mass spectrometry data of individual peptides was searched with SEQUEST. Using this approach we demonstrated a successful proof-of-concept experiment by identifying TMV proteins present in the total protein extract. The same procedure was then applied to tobacco plants infected with a laboratory viral isolate of unknown identity. Several of the differentially expressed protein spots were identified as proteins of potato virus X (PVX), thus successfully identifying the causative agent of the uncharacterized viral infection. We believe this demonstrates that HPLC-MS/MS can be used to successfully characterize unknown viruses in infected plants. (J Am Soc Mass Spectrom 2003, 14, 736 -741)

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A polymerase chain reaction method adapted for selective amplification and cloning of 3' sequences of potyviral genomes: application to dasheen mosaic virus

Cited by 133 publications

References 39 publications

Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus

Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus

Caracterização do gene da proteína capsidial do Grapevine virus A em videiras afetadas pela acanaladura do lenho de Kober no Estado de São Paulo

Investigative proteomics: Identification of an unknown plant virus from infected plants using mass spectrometry

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