Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections

Phylogenetic analysis of Tomato mosaic virus from Hemerocallis sp. and Impatiens hawkeri Análise filogenética de Tomato mosaic virus isolado de Hemerocallis sp. e Impatiens hawkeri

The culture and commercialization of ornamental plants have considerably increased in the last years. To supply the commercial demand, several Hemerocallis and Impatiens varieties have been bred for appreciated qualities such as flowers with a diversity of shapes and colors. With the aim of characterizing the tobamovirus isolated from Hemerocallis sp. (tobamo-H) and Impatiens hawkeri (tobamo-I) from the USA and São Paulo, respectively, as well as to establish phylogenetic relationships between them and other Tobamovirus species, the viruses were submitted to RNA extraction, RT-PCR amplification, coat-protein gene sequencing and phylogenetic analyses. Comparison of tobamovirus homologous sequences yielded values superior to 98.5% of identity with Tomato mosaic virus (ToMV) isolates at the nucleotide level. In relation to tobamo-H, 100% of identity with ToMV from tomatoes from Australia and Peru was found. Based on maximum likelihood (ML) analysis it was suggested that tobamo-H and tobamo-I share a common ancestor with ToMV, Tobacco mosaic virus, Odontoglossum ringspot virus and Pepper mild mottle virus. The tree topology reconstructed under ML methodology shows a monophyletic group, supported by 100% of bootstrap, consisting of various ToMV isolates from different hosts, including some ornamentals, from different geographical locations. The results indicate that Hemerocallis sp. and I. hawkeri are infected by ToMV. This is the first report of the occurrence of this virus in ornamental species in Brazil.<br>O cultivo e comercialização de plantas ornamentais têm aumentado consideravelmente nos últimos anos. Para suprir a demanda comercial, diversas variedades de Hemerocallis sp. e Impatiens hawkeri têm sido desenvolvidas pelas qualidades apreciáveis como flores com diversidade de formas e cores. Com o objetivo de caracterizar o tobamovirus isolado de Hemerocallis sp. (tobamo-H) e Impatiens hawkeri (tobamo-I) provenientes dos EUA e São Paulo, respectivamente, assim como estabelecer relações filogenéticas entre os isolados e outras espécies de Tobamovirus, foram realizados extrações do RNA, RT-PCR, seqüenciamento do gene da capa protéica e análises filogenéticas. Quando foram comparadas seqüências homólogas de tobamovirus, valores superiores a 98,5% de identidade, ao nível de nucleotídeos, foram obtidos com isolados do Tomato mosaic virus (ToMV). Em relação ao tobamo-H, foi encontrado 100% de identidade com o ToMV isolado de tomateiro da Austrália e do Peru. Com base em análise de máxima verossimilhança (MV), sugere-se que o tobamo-H e tobamo-I compartilham um ancestral comum com ToMV, Tobacco mosaic virus, Odontoglossum ringspot virus e Pepper mild mottle virus. A topologia da árvore reconstruída a partir de MV mostra um grupo monofilético, sustentado por 100% de "bootstrap", formado por vários isolados de ToMV de diferentes hospedeiras, incluindo ornamentais, a partir de diferentes localizações geográficas. Os resultados indicam que Hemerocallis sp.e I. hawkeri estão infectados pelo ToMV, sendo esse o primeiro relato desse vírus em ornamentais, no Brasil