Import of plasmid DNA into the nucleus is sequence specific
Import of plasmid DNA into the nucleus is sequence specific
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DOI：
10.1006/excr.1996.3427
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发表时间：
1997-02-01
影响因子：
3.7
通讯作者：
Dean, DA
中科院分区：
文献类型：
--
作者：
Dean, DA
在非分裂细胞中，质粒DNA的核输入是许多病毒生命周期、基因治疗方案和基因表达实验成功的重要过程。在这里，完整的无蛋白SV40 DNA被细胞质注入细胞，其亚细胞定位随后进行原位杂交。定位于细胞核的SV40 DNA与通过核孔复合物（NPC）的运输机制一致：添加NPC抑制剂小麦胚芽凝集素和抗核孔蛋白抗体以及能量消耗可以抑制其进口。DNA的转运似乎是一个多步骤的过程，DNA在其输入之前在核周围积累。最重要的是，核输入是序列特异性的：SV40 DNA的一个包含复制起源和早期和晚期启动子的区域支持输入，而细菌序列和其他SV40衍生序列不支持输入。大多数输入的DNA与SC-35剪接复合体抗原共定位，表明核内DNA定位于转录或信息处理区域。这种与转录的联系通过发现抑制转录阻断DNA输入而不是蛋白质核输入而得到加强。综上所述，这些结果支持一种模型，即质粒DNA核输入的机制类似于含有核定位信号的蛋白质，但也依赖于转录。(C) 1997学术出版社。
Nuclear import of plasmid DNA in nondividing cells is a process essential to the success of numerous viral life cycles, gene therapy protocols, and gene expression experiments, Here, intact protein-free SV40 DNA was cytoplasmically injected into cells and its subcellular localization was followed by in situ hybridization. SV40 DNA localized to the nucleus consistent with a mechanism of transport through the nuclear pore complex (NPC): import was inhibited by the addition of the NPC-inhibitory agents wheat germ agglutinin and an anti-nucleoporin antibody as well as by energy depletion. DNA transport appeared to be a multistep process with the DNA accumulating at the nuclear periphery before its import. Most importantly, nuclear import was sequence specific: a region of SV40 DNA containing the origin of replication and the early and late promoters supported import, whereas bacterial sequences alone and other SV40-derived sequences did not. The majority of the imported DNA colocalized with the SC-35 splicing complex antigen, suggesting that the intranuclear DNA localizes to areas of transcription or message processing. This link to transcription was strengthened by the finding that inhibition of transcription blocked DNA import but not protein nuclear import. Taken together, these results support a model in which plasmid DNA nuclear import occurs by a mechanism similar to that used by nuclear localization signal-containing proteins but is also dependent on transcription. (C) 1997 Academic Press.