Goodier John L, Ostertag Eric M, Engleka Kurt A, Seleme Maria C, Kazazian Haig H
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Hum Mol Genet. 2004 May 15;13(10):1041-8. doi: 10.1093/hmg/ddh118. Epub 2004 Mar 17.
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.
由于在检测该蛋白的内源性或过表达形式方面存在技术限制，迄今为止，确定L1开放阅读框2蛋白（ORF2p）的亚细胞定位是不可能的。在此，我们报告了在改良痘苗病毒/T7 RNA聚合酶（MVA/T7RP）系统中表达后，在培养的人类细胞中全长ORF2p的可视化情况。MVA/T7RP系统用于确定L1 ORF1p和ORF2p作为与绿色荧光蛋白的融合体以及通过免疫细胞化学的亚细胞定位。全长ORF2p主要位于细胞质中，而羧基末端缺失的ORF2p还定位于核仁。我们在ORF2p中定位了一个功能性核仁定位信号。ORF1p出现在细胞质中，呈斑点状模式，并在一部分细胞的核仁中与ORF2p共定位。这些发现有助于解释最近在人类基因组中发现的L1与小RNA基因序列之间嵌合体的存在。
