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The grass goldfish appeared early in the evolutionary history of goldfish, and shows heritable stability in the development of the caudal fin. The twin-tail phenotype is extremely rare, however, some twin-tail individuals were produced in the process of breeding for ornamental value. From mutations in the twin-tail goldfish genome, we identified two kinds of Tgf2 transposons. One type was completely sequenced Tgf2 and the other type was ΔTgf2, which had 858 bp missing. We speculate that the bifurcation of the axial skeletal system in goldfish may be caused by an endogenous ΔTgf2 insertion mutation in Chordin A, as ΔTgf2 has no transposition activity and blocks the expression of Chordin A. The twin-tail showed doubled caudal fin and accumulation of red blood cells in the tail. In addition, in situ hybridization revealed that ventral embryonic tissue markers (eve1, sizzled, and bmp4) were more widely and strongly expressed in the twin-tail than in the wild-type embryos during the gastrula stage, and bmp4 showed bifurcated expression patterns in the posterior region of the twin-tail embryos. These results provide new insights into the artificial breeding of genetically stable twin-tail grass goldfish families.

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., (Copyright © 2016. Published by Elsevier B.V.)

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.

Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.

The goldfish (Carassius auratus) Tgf2 transposon is a vertebrate DNA transposon that belongs to the hAT transposon family. In this study, we constructed plasmids containing either the full-length Tgf2 transposon (pTgf2 plasmid) or a partially-deleted Tgf2 transposon (ΔpTgf2 plasmid), and microinjected these plasmids into fertilized zebrafish (Danio rerio) eggs at the one- to two-cell stage. DNA extracted from the embryos was analyzed by PCR to assess transient excision, if any, of the exogenous plasmid and to verify whether Tgf2 is an autonomous transposon. The results showed that excision-specific bands were not detected in embryos injected with the ΔpTgf2 plasmid, while bands of 300-500bp were detected in embryos injected with pTgf2, which indicated that the full-length Tgf2-containing plasmid could undergo autonomous excision in zebrafish embryos. DNA cloned from 24 embryos injected with pTgf2 was sequenced, and the results suggested that Tgf2 underwent self-excision in zebrafish embryos. Cloning and PCR analysis of DNA extracted from embryos co-injected with ΔpTgf2 and in vitro-transcribed transposase mRNA indicated that partially-deleted-Tgf2-containing ΔpTgf2 plasmid also underwent excision, in the presence of functional transposase mRNA. DNA cloned from 25 embryos co-injected with ΔpTgf2 and transposase mRNA was sequenced, and the results suggested that partially-deleted Tgf2 transposons plasmids were excised. These results demonstrated that excisions of Tgf2 transposons were mediated by the Tgf2 transposase, which in turn confirmed that Tgf2 is an autonomous transposon., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hobo/Activator/Tam3 (hAT) superfamily transposons occur in plants and animals and play a role in genomic evolution. Certain hAT transposons are active and have been developed as incisive genetic tools. Active vertebrate elements are rarely discovered; however, Tgf2 transposon was recently discovered in goldfish (Carassius auratus). Here, we found that the endogenous Tgf2 element can transpose in goldfish genome. Seven different goldfish mRNA transcripts, encoding three lengths of Tgf2 transposase, were identified. Tgf2 transposase mRNA was detected in goldfish embryos, mainly in epithelial cells; levels were high in ovaries and mature eggs and in all adult tissues tested. Endogenous Tgf2 transposase mRNA is active in mature eggs and can mediate high rates of transposition (>30%) when injected with donor plasmids harboring a Tgf2 cis-element. When donor plasmid was coinjected with capped Tgf2 transposase mRNA, the insertion rate reached >90% at 1 yr. Nonautonomous copies of the Tgf2 transposon with large-fragment deletions and low levels of point mutations were also detected in common goldfish. Phylogenetic analysis indicates the taxonomic distribution of Tgf2 in goldfish is not due to vertical inheritance. We propose that the goldfish Tgf2 transposon originated by recent horizontal transfer and maintains a highly native activity.

The hAT transposon family, including hobo of Drosophila, Ac of maize (Zea mays L.) and Tam3 of snapdragon (Ceratophyllum demersum L.), is proposed to be involved in transposition between genomic DNAs in "cut and paste" patterns. In 1996, a transposon of Tol2, the first autonomous transposon in vertebrate, was identified from the genome of albino medaka fish (Oryzias latipes). Since then, a new transgenic and gene trap system based on Tol2 has been developed and widely used in zebrafish. In this study, we designed gene-specific primers based on the conserved regions of amino acid sequences between medaka Tol2 and maize Ac. Meanwhile, PCR was carried out in 19 fish species or strains including goldfish. Finally, another similar hAT transposon, termed as Tgf2, was identified in the genomes from different strains of goldfish. Goldfish Tgf2 is 4720 bp in length including 4 exons and shares an identity of 97% with medaka Tol2. Distinct differences were observed in the terminal inverted repeat (TIR) and subterminal repeat (STR) regions between Tgf2 and Tol2. In addition, the internal inverted repeat (IIR) region of Tgf2 (1453 bp-2091 bp) tended to form a "+" crossing structure, rather than a stem-loop structure in medaka Tol2. The regions, including TIR, STR, and IIR, were supposed to be closely related to the transposing activities of hAT transposon family members. From these sequence differences, we may expect a probably high activity of goldfish Tgf2 in transposition and it could be further used as a tool for transgenesis and gene trap in aquaculture fish in future.