Meller V H, McConnell M, Fisher P A
Department of Pharmacological Sciences, University Medical Center, State University of New York at Stony Brook 11794-8651.
J Cell Biol. 1994 Sep;126(6):1331-40. doi: 10.1083/jcb.126.6.1331.
Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).
在0至2小时大的果蝇胚胎的无细胞提取物中，大多数DNA拓扑异构酶II（拓扑异构酶II）似乎是非核的，在低速离心（10,000g）后仍留在上清液中。实际上，所有这种明显可溶的拓扑异构酶II都是颗粒状的，沉降系数为67S。在果蝇Kc组织培养细胞、16至19小时大的胚胎以及非洲爪蟾孕酮成熟卵母细胞的提取物中也检测到了类似的含拓扑异构酶II的颗粒。果蝇含拓扑异构酶II的颗粒对EDTA、Triton X-100和DNase I不敏感，但可通过与0.3M NaCl或RNase A孵育而被破坏。经过任何一种破坏处理后，拓扑异构酶II以9S沉降。含拓扑异构酶II的颗粒对微球菌核酸酶也敏感。化学交联的结果证实了通过离心获得的结果。免疫印迹分析表明，含拓扑异构酶II的颗粒缺乏大量的核纤层蛋白、核孔复合体蛋白gp210、增殖细胞核抗原、RNA聚合酶II亚基、组蛋白、卷曲螺旋蛋白和核仁蛋白。Northern印迹分析表明，含拓扑异构酶II的颗粒缺乏U RNA。因此，目前的数据支持这样一种观点，即果蝇非核含拓扑异构酶II的颗粒主要由拓扑异构酶II和一种未知的RNA分子组成。
