Fedorov Anton, Lukyanov Dmitri, Rogoliński Jacek, Widłak Piotr, Podgornaya Olga, Rzeszowska-Wolny Joanna
Institute of Cytology, Russian Academy of Science, Tikhoretsky pr. 4, 194064 St. Petersburg, Russia.
Cell Mol Biol Lett. 2004;9(1):153-65.
In our previous study, a 454 bp DNA fragment was isolated from rat genomic DNA as an element which interacts with nuclear matrix proteins, i.e. a Matrix Associated Region (MAR). Computer analyses revealed that the right half of this fragment, named RME (Rat MAR Element), possesses a high matrix association potential and is likely to be responsible for the matrix association of the whole sequence. RME was used as a probe in an electrophoretic mobility shift assay (EMSA), and with the use of Southwestern blotting, a rat liver nuclear protein which binds specifically to it was identified. Its molecular mass was estimated by SDS-PAGE as 30 kDa (p30). Polyclonal antibodies raised against protein-RME complexes caused a super-shift of specific complexes in EMSA, and bound to p30 in nuclear extracts of rat liver in Western blotting. The immunofluorescence labelling of a rat embryonic fibroblast cell monolayer with anti-p30 antibody revealed a mainly intranuclear pattern of staining.
在我们之前的研究中，从大鼠基因组DNA中分离出一个454 bp的DNA片段，作为与核基质蛋白相互作用的元件，即基质相关区域（MAR）。计算机分析表明，该片段的右半部分，命名为RME（大鼠MAR元件），具有较高的基质结合潜力，可能负责整个序列与基质的结合。RME用作电泳迁移率变动分析（EMSA）中的探针，并通过蛋白质印迹法，鉴定出一种与之特异性结合的大鼠肝核蛋白。通过SDS-PAGE估计其分子量为30 kDa（p30）。针对蛋白质-RME复合物产生的多克隆抗体在EMSA中导致特异性复合物超迁移，并在蛋白质印迹法中与大鼠肝核提取物中的p30结合。用抗p30抗体对大鼠胚胎成纤维细胞单层进行免疫荧光标记，显示主要为核内染色模式。
