Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug

CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOHeterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA3/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation. © 2011 Elsevier Ltd.Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA3/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.426568578CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO47303/2009-72010-50015-6132341/2010-7503702/2007-2301943/2009-5Adams, M.D., The genome sequence of Drosophila melanogaster (2000) Science, 287, pp. 2185-2195Allis, C.D., Jenuwein, T., Reinberg, D., Caparros, M.L., (2007) Epigenetics, , Cold Spring Harbor Lab. 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