Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Cell Culture Techniques
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Cell Nucleolus / chemistry*
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Cell Nucleolus / ultrastructure
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Cloning, Molecular
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Cytoplasm / ultrastructure
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DNA Transposable Elements
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DNA-Directed RNA Polymerases / genetics
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Endonucleases / analysis*
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Endonucleases / genetics
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Endonucleases / metabolism
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Endothelial Cells / ultrastructure
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Green Fluorescent Proteins / analysis
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Green Fluorescent Proteins / genetics
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Humans
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Immunochemistry
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Long Interspersed Nucleotide Elements / genetics*
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Nuclear Localization Signals / genetics
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RNA-Directed DNA Polymerase / analysis*
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RNA-Directed DNA Polymerase / genetics
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RNA-Directed DNA Polymerase / metabolism
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Ribonucleoproteins / analysis*
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Ribonucleoproteins / genetics
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Ribonucleoproteins / metabolism
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Umbilical Veins / cytology
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Viral Proteins
Substances
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DNA Transposable Elements
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L1 ORF1 protein, human
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L1 ORF2 protein, human
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Nuclear Localization Signals
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Ribonucleoproteins
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Viral Proteins
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enhanced green fluorescent protein
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Green Fluorescent Proteins
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bacteriophage T7 RNA polymerase
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RNA-Directed DNA Polymerase
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DNA-Directed RNA Polymerases
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Endonucleases