Ma H, Siegel A J, Berezney R
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260, USA.
J Cell Biol. 1999 Aug 9;146(3):531-42. doi: 10.1083/jcb.146.3.531.
To study the possible role of the nuclear matrix in chromosome territory organization, normal human fibroblast cells are treated in situ via classic isolation procedures for nuclear matrix in the absence of nuclease (e.g., DNase I) digestion, followed by chromosome painting. We report for the first time that chromosome territories are maintained intact on the nuclear matrix. In contrast, complete extraction of the internal nuclear matrix components with RNase treatment followed by 2 M NaCl results in the disruption of higher order chromosome territory architecture. Correlative with territorial disruption is the formation of a faint DNA halo surrounding the nuclear lamina and a dispersive effect on the characteristically discrete DNA replication sites in the nuclear interior. Identical results were obtained using eight different human chromosome paints. Based on these findings, we developed a fractionation strategy to release the bulk of nuclear matrix proteins under conditions where the chromosome territories are maintained intact. A second treatment results in disruption of the chromosome territories in conjunction with the release of a small subset of acidic proteins. These proteins are distinct from the major nuclear matrix proteins and may be involved in mediating chromosome territory organization.
为研究核基质在染色体领地组织中的可能作用，采用经典的核基质分离方法，在无核酸酶（如脱氧核糖核酸酶I）消化的情况下对正常人成纤维细胞进行原位处理，随后进行染色体涂染。我们首次报道染色体领地在核基质上保持完整。相反，先用核糖核酸酶处理，再用2M氯化钠处理以完全提取核基质内部成分，会导致高阶染色体领地结构的破坏。与领地破坏相关的是在核纤层周围形成微弱的DNA晕圈，以及对核内特征性离散DNA复制位点的分散作用。使用八种不同的人类染色体涂染剂获得了相同的结果。基于这些发现，我们制定了一种分级分离策略，在染色体领地保持完整的条件下释放大部分核基质蛋白。第二次处理会导致染色体领地的破坏，并释放一小部分酸性蛋白。这些蛋白质与主要的核基质蛋白不同，可能参与介导染色体领地的组织。
