Department of Anatomy, Cell Biology and Physiology, Institute of Biology, University of Campinas (UNICAMP), 13083-863 Campinas, SP, Brazil.
Micron. 2011 Aug;42(6):568-78. doi: 10.1016/j.micron.2011.02.002. Epub 2011 Feb 16.
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 CMA(3)/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.
锥蝽，即恰加斯病的传播媒介，其单中心和多中心间期细胞核中的异染色质体曾被认为含有富含 AT 的 DNA，这是基于它们对 Q 带和 Hoechst 33248 处理的阳性反应。目前尚不清楚这些核中是否也存在富含 GC 的 DNA，以及它是否在染色质浓缩中发挥作用。考虑到目前可获得比以前用于原位测定 DNA 碱基组成的方法更精确的方法，并且不同物种的间期细胞核中组成不同的染色质区域的空间分布是一个有趣的问题，因此重新研究了富含 AT 和 GC 的 DNA 在 T. infestans 核中的定位。所使用的方法包括 DAPI/AMD 和 CMA(3)/Distamycin 差异染色、Msp I 和 Hpa II 酶消化后 Feulgen-DNA 图像分析、5-甲基胞嘧啶免疫检测、AgNOR 反应、共聚焦显微镜以及 5-氮杂-2'-脱氧胞苷 (5-AZA) 去甲基化测定。结果表明，染色质中心存在富含 AT/GC 贫 DNA，常染色质中均匀分布着 AT 和 GC 序列。还发现了一个环绕染色质中心的富含 GC 的 DNA 区，但它不能与 NOR 区域相关联。为了证实 T. infestans 核中富含 DNA 的 AT，还进行了生物信息学分析。在单中心和多中心核的染色质体边缘以及多中心核的常染色质体的某些点上，可以明显看到单甲基化胞嘧啶，并且可以暂时受到 5-AZA 处理的影响。目前的结果表明，在半翅目 T. infestans 的染色质体的特殊情况下，胞嘧啶甲基化不是参与染色质浓缩的相关因素。
