title: Identification of nuclear localization signal within goldfish Tgf2 transposase.
abstract: The structure of goldfish (Carassius auratus) Tgf2 transposase is still poorly understood, although it can mediate efficient gene transfer in teleost fish. We hypothesized the existence of a nuclear localization signal (NLS) within Tgf2 transposase to assist transport into the nucleus. To explore this, 15 consecutive amino acid residues (656-670 aa) within the C-terminus of Tgf2 transposase were predicted in silico to be a NLS domain. The pEGFP-C1-Tgf2TP(△31C) plasmid encoding the NLS-domain-deleted Tgf2 transposase fused to EGFP was constructed, and transfected into 293T cells. After transfection with pEGFP-C1-Tgf2TP(△31C), EGFP was not detected in the nucleus alone, while 67.0% of cells expressed EGFP only in the cytoplasm. In contrast, after transfection with control plasmids containing C- or N-terminal truncated Tgf2 transposases with an intact NLS domain, EGFP was not detected in the cytoplasm alone, while approximately 40% of cells expressed EGFP only in the nucleus, and the remaining 60% expressed EGFP in both the nucleus and cytoplasm. Our results demonstrated that loss of the NLS domain results in expression in the cytoplasm but not in the nucleus. These findings suggest that 15 aa residues located from 656 to 670 aa within the C-terminus of Tgf2 transposase can function as a NLS to assist the transfer of the transposase into the nucleus where it mediates DNA transposition.
authors.affiliation: Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China.
authors.forename: Xiao-Dan
authors.initials: XD
authors.lastname: Shen
authors.affiliation: Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Hou
authors.affiliation: Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China. Electronic address: jiangxy@shou.edu.cn.
authors.forename: Xia-Yun
authors.initials: XY
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China. Electronic address: smzou@shou.edu.cn.
authors.forename: Shu-Ming
authors.initials: SM
authors.lastname: Zou
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D006054:Goldfish / Q000235:genetics / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D019895:Transposases / Q000235:genetics / Q000378:metabolism
chemical_list: D029941:Fish Proteins; D019913:Nuclear Localization Signals; D019895:Transposases
keywords: Cell culture; DNA transposon; Nuclear localization signal; Tgf2 transposase