title: The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.
abstract: Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition.
authors.affiliation: College of Food Science and Technology, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.|Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors.forename: Xia-Yun
authors.initials: XY
authors.lastname: Jiang
authors.affiliation: College of Food Science and Technology, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Hou
authors.affiliation: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors.forename: Xiao-Dan
authors.initials: XD
authors.lastname: Shen
authors.affiliation: College of animal science and technology, Yangzhou University, Wenhui Road 48, Yangzhou 225009, China.
authors.forename: Xue-Di
authors.initials: XD
authors.lastname: Du
authors.affiliation: College of Food Science and Technology, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors.forename: Hai-Li
authors.initials: HL
authors.lastname: Xu
authors.affiliation: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors.forename: Shu-Ming
authors.initials: SM
authors.lastname: Zou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004251:DNA Transposable Elements; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D006054:Goldfish / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008958:Models, Molecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D019895:Transposases / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish; D016335:Zinc Fingers
chemical_list: D004251:DNA Transposable Elements; D029941:Fish Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; D019895:Transposases