Goldfish transposaseTgf2presumably from recent horizontal transfer is active

Abstract

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.

Prokaryotic Expression and Purification of Soluble Goldfish Tgf2 Transposase with Transposition Activity

“…The donor plasmid of pTgf2-EF1a-EGFP containing 220 and 185 bp of the left and right ends of the goldfish Tgf2 transposon was previously constructed [19]. The microinjection volume was estimated at 1 nl/embryo.…”

“…The microinjection volume was estimated at 1 nl/embryo. And 50 pg circular donor plasmid pTgf2-EF1a-EGFP was injected alone, or co-injected with 2 ng recombinant Tgf2 TPase protein, into blunt snout bream embryos at the 1-2 cell stage [19]. After injection, embryos were placed in embryo rearing medium and maintained at room temperature.…”

“…PCR amplifications were performed using genomic DNA from tail fin of blunt snout bream. Cloning and sequencing were conducted as previously described [19].…”

“…The goldfish Tgf2 transposon is 4,720 bp in length and consists of four exons, which can transcribe a complete 2,633 bp cDNA encoding a 686 a.a. Tgf2 TPase [19]. The 2061-bp Tgf2 TPase ORF was cloned into pET-28a(?)…”

“…Tgf2 from goldfish (Carassius auratus) is an autonomous hAT transposon in vertebrate, and encodes an intact and active transposase [19,20]. Several studies previously demonstrated that the Tgf2 transposase (Tgf2 TPase) produced by translation of microinjected mRNA could mediate gene transposition in aquacultural fish species when embryos were co-injected with a donor nonautonomous plasmid harboring the Tgf2 cis-element [21][22][23].…”