Abstract
Here we report a general system, inducible translocation trap (ITT), for identification of proteins that translocate into the nucleus following signal transduction from cell surface receptors. ITT consists of a retroviral cDNA expression library of fusion proteins consisting of a LexA DNA binding domain, the transactivation domain of a transcriptional activator, and proteins encoded by cDNA inserts. The retroviral library is then transduced into cell lines containing a reporter gene with LexA binding sites in its promoter. Cells expressing the reporter gene by extracellular stimuli are then selected by flow-cytometric sorting. By using ITT, we identified cDNA encoding Stat1 in a screen of proteins which translocate into the nucleus by IFNgamma, indicating that this system can be used for isolation of nuclear translocating proteins induced by extracellular stimuli. ITT may be a useful tool for dissecting dynamic translocation in various biological systems.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Active Transport, Cell Nucleus / genetics*
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Animals
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Bacterial Proteins / genetics
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Binding Sites / genetics
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Cell Line
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DNA, Complementary / analysis*
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DNA, Complementary / genetics
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DNA-Binding Proteins / genetics
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Flow Cytometry
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Genes, Reporter / genetics*
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Genetic Vectors / genetics*
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Genetic Vectors / metabolism
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Genomic Library
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Interferon-gamma / genetics
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Interferon-gamma / metabolism
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Mice
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Promoter Regions, Genetic / genetics
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Protein Structure, Tertiary / genetics
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Protein Transport / genetics*
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Recombinant Fusion Proteins / genetics*
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Recombinant Fusion Proteins / metabolism
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Reproducibility of Results
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Retroviridae / genetics
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STAT1 Transcription Factor
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Serine Endopeptidases / genetics
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Trans-Activators / genetics
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Transduction, Genetic / methods*
Substances
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Bacterial Proteins
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DNA, Complementary
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DNA-Binding Proteins
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LexA protein, Bacteria
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Recombinant Fusion Proteins
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STAT1 Transcription Factor
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Stat1 protein, mouse
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Trans-Activators
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Interferon-gamma
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Serine Endopeptidases