Urasaki, A., Morvan, G., and Kawakami, K. (2006) Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics. 174(2):639-649.
The Tol2 element is a naturally occurring active transposable element found from a vertebrate genome. 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 (5') and the right (3')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 more than 10-kb could transpose, but a certain length of spacer, less than 276-bp but more than 18-bp, between the left and right ends was necessary for excision. Furthermore, we found a 5-bp sequence, 5'-(A/G)AGTA-3', 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 to understand roles of the repetitive sequence in the subterminal region in transposition.