Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences
Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences
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DOI：
10.1074/jbc.274.31.22025
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发表时间：
1999-07-30
影响因子：
4.8
通讯作者：
Dean, DA
中科院分区：
文献类型：
--
作者：
Wilson, GL;Dean, BS;Dean, DA
虽然对信号介导的蛋白质和RNA核输入和输出的机制了解很多，但对质粒DNA的核输入了解很少，使用荧光素缀合的肽核酸钳特异性标记4.2和14.4之间的质粒DNA酶。所得到的基板能够在细胞分裂的情况下，在显微注射的细胞中的基因表达和核定位。为了阐明质粒核输入的要求，采用毛地黄皂苷透化的细胞系统来跟踪质粒的核定位。标记质粒的核输入具有时间和能量依赖性，并受外源凝集素麦胚凝集素的抑制，对胞质提取物有绝对的需求。单独添加核提取物不支持质粒核输入，但与细胞质组合刺激质粒核定位。尽管添加纯化的输入蛋白α、输入蛋白β和RAN足以支持蛋白质核输入，但质粒核输入还需要添加核提取物。最后，质粒DNA的核输入是序列特异性的，需要SV40早期启动子和增强子的区域。总之，这些结果证实并扩展了我们在显微注射细胞中的发现，并支持蛋白质介导的质粒核输入机制。
Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA, Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluorescein-conjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time- and energy-dependent, was inhibited by the lectin wheat germ ag glutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin alpha, importin beta, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import.