Functional Dissection of the Tol2 Transposable Element Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in the Subterminal Region Essential for Transposition

Abstract

The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been shown to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (59) and the right (39) ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of .11 kb could transpose, but a certain length of spacer, ,276 but .18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 59-(A/G)AGTA-39, is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.

Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish

“…1 A-C), T2MUASTeTxLC, and T2SUASTeTxLCCFP (Figs. 3A and 4A) were constructed based on Tol2 vectors, T2KXIG (13), T2AL200R150G, T2KXIG⌬in (29), and T2MUASMCS, which contains a synthetic multicloning site downstream of five tandem repeats of the Gal4 binding sequence (5xUAS) and a TATA sequence. The Gal4FF gene (or referred to as GFF) is a fusion of the DNA binding domain from the Gal4 protein and two transcription activation modules (2xPADALDDFDLDML) from VP16.…”

“…For each cross, at least 40 F 1 embryos were analyzed. The integration sites of the transposon insertions were analyzed by inverse PCR and linker-mediated PCR (29,33,34). These transgenic fish are designated as hspGGFF, hspGFF, and SAGFF, respectively.…”

Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase

“…Briefly, the mCherry gene was cloned at BamHI and ClaI sites of the pT2AL200R150G vector, 47 replacing the EGFP gene (pT2-mCherry). Total RNA from whole zebrafish embryos was prepared using the Trizol Reagent.…”

Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro

“…Thus in this study, we used Bmal1-promoter-and Dbp-promoter-driven luciferase reporters to visualize the intrinsic cellular circadian clock. We cotransfected ES cells with Tol2 transposase (TP) expression plasmid and Tol2 transposon-based Bmal1:luc or Dbp:luc reporter vectors (Bmal1:luc-pT2A, Dbp:luc-pT2A) (13,14). The Tol2 transposon was originally discovered in Medaka fish, and the Tol2-based vector is considered a highly efficient gene transfer system in mouse ES cells (13,15).…”