A potential role for the nucleolus in L1 retrotransposition
A potential role for the nucleolus in L1 retrotransposition
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DOI：
10.1093/hmg/ddh118
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发表时间：
2004-05-15
影响因子：
3.5
通讯作者：
Kazazian, HH
中科院分区：
文献类型：
--
作者：
Goodier, JL;Ostertag, EM;Kazazian, HH
由于检测L1 ORF2蛋白(ORF2p)内源性或过度表达形式的技术限制，迄今无法确定该蛋白的亚细胞定位。在这里，我们报告了在改良的痘苗病毒/T7RNA聚合酶(MVA/T7RP)系统中表达的全长ORF2p在培养的人类细胞中的可视化。MVA/T7RP系统用于确定L1ORF1p和ORF2p作为绿色荧光蛋白融合蛋白的亚细胞定位和免疫细胞化学。全长ORF2p以胞质为主，而羧基末端缺失的ORF2p定位于核仁。我们在ORF2p中定位了一个功能核仁定位信号。ORF1p以斑点状出现在细胞质中，并与ORF2p共存于部分细胞的核仁中。这些发现有助于解释为什么最近在人类基因组中发现的L1s和小RNA基因序列之间存在嵌合体。
Determining the subcellular localization of the L1 ORF2 protein (ORF2p) has been impossible to date because of technical limitations in detecting either endogenous or overexpressed forms of the protein. Here we report visualization of the full-length ORF2p in cultured human cells following expression in a modified vaccinia virus/T7 RNA polymerase (MVA/T7RP) system. The MVA/T7RP system was used to ascertain subcellular localization of L1 ORF1p and ORF2p both as fusions with green fluorescent protein and by immunocytochemistry. Full-length ORF2p was predominantly cytoplasmic, while carboxy-terminal-deleted ORF2p localized additionally to the nucleolus. We mapped a functional nucleolar localization signal in ORF2p. ORF1p appeared in the cytoplasm with a speckled pattern and colocalized with ORF2p in nucleoli in a subset of cells. These findings help explain the presence of chimeras between L1s and small RNA gene sequences recently discovered in the human genome.