RESUMO
The nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) from leaves of Drosera (Droseraceae) were amplified using "universal" primers. The analysis of the products demonstrated most samples were a molecular mixture as a result of unsuccessful and non-specific amplifications. Among the obtained sequences, two were from Basidiomycota fungi. Homologous sequences of Basidiomycota were obtained from GenBank database and added to a data set with sequences from Drosera leaves. Parsimony analysis demonstrated that one sequence was amplified from an Ustilaginomycetes fungus, and another from a Heterobasidiomycetes. Possibly these fungi were associated to leaves of Drosera, and not because of samples contamination. In order to provide optimization and a better specificity of PCR (polymerase chain reaction), a very successful method was demonstrated using dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) in reactions.
Os espaçadores internos transcritos do DNA nuclear ribossomal (ITS1 e ITS2) de folhas de Drosera (Droseraceae) foram amplificados com o emprego de iniciadores "universais". A análise demonstrou que a maior parte das amostras continha uma mistura resultante de amplificações não-específicas. Dentre as sequências de DNA obtidas, duas delas foram de fungos basidiomicetos. Sequências homólogas foram obtidas do GenBank e analisadas junto às sequências obtidas de folhas de Drosera. Através das análises filogenéticas de máxima parcimônia foi possível identificar uma seqüência como sendo de um Ustilaginomycetes e outra de Heterobasidiomycetes (Basidiomycota). Possivelmente esses organismos estavam associados às folhas de Drosera e assim não sejam resultantes de contaminação. Com o objetivo de otimizar e buscar uma melhor especificidade das reações de PCR, um protocolo bem sucedido foi demonstrado com o uso de dimetilsulfóxido (DMSO) e soroalbumina bovina (BSA).
RESUMO
The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvoviruses. In spite of the high sequence similarity, genetic analysis has shown the existence of at least two virus lineages among the samples. In conclusion, these results highlight the need for close surveillance on PPV genetic drift, with an assessment of its potential ability to modify the antigenic make-up of the virus.