Green fluorescent protein tag for studies of drug-induced translocation of nucleolar protein RH-II/Gu.
Green fluorescent protein tag for studies of drug-induced translocation of nucleolar protein RH-II/Gu.
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DOI：
10.2144/98246cr03
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发表时间：
1998-06
期刊：
影响因子：
2.7
通讯作者：
B. Valdez;L. Perlaky;Z. Cai;D. Henning;H. Busch
中科院分区：
文献类型：
--
作者：
B. Valdez;L. Perlaky;Z. Cai;D. Henning;H. Busch
我们构建了一个人成骨肉瘤细胞系，U-2 OS/GFP-Gu，表达核仁RNA解旋酶RH-II/Gu标记的绿色荧光蛋白（GFP）。GFP标签的存在不抑制RH-II/Gu蛋白的RNA解旋酶、RNA折叠和ATP酶活性。衍生的细胞系响应于细胞毒性剂，如亲本细胞系U-2 OS。在放线菌素D或丰霉素的存在下，GFP-RH-II/Gu融合蛋白以与内源性RH-II/Gu易位相同的方式从核仁易位到核质。药物诱导的GFP-RH-II/Gu易位很容易通过直接观察体内活细胞来监测。该细胞系可用于筛选细胞毒药物和研究药物诱导的RH-II/Gu易位机制。在细胞周期依赖性的核仁形成过程中，RH-II/Gu的细胞定位很容易监测。更快地获得实时结果，而没有与细胞固定和基于免疫荧光的染色相关的缺点。
We have constructed a human osteogenic sarcoma cell line, U-2 OS/GFP-Gu, that expresses nucleolar RNA helicase RH-II/Gu tagged with green fluorescent protein (GFP). The presence of a GFP tag does not inhibit RNA helicase, RNA folding and ATPase activities of RH-II/Gu protein. The derived cell line responds to cytotoxic agents like the parental cell line U-2 OS. In the presence of either actinomycin D or toyocamycin, the GFP-RH-II/Gu fusion protein translocates from the nucleolus to the nucleoplasm in the same way as the translocation of endogenous RH-II/Gu. The drug-induced translocation of GFP-RH-II/Gu is easily monitored by direct observation of live cells in vivo. This cell line can be used to screen cytotoxic drugs and to study the mechanisms of drug-induced translocation of RH-II/Gu. The cellular localization of RH-II/Gu during the cell cycle-dependent formation of the nucleolus is readily monitored. Real-time results are obtained more quickly without the disadvantages associated with cell fixation and immunofluorescence-based staining.