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Materials and methods (Supplement) Construction of a expression plasmid The coding region of the Tol2 transposase gene Tol2TP (AB032244, 1782 bp) was amplified by PCR with primers Tol2TP-F (5’– cg ggatcc ACCAT GGAGG AAGTA TGTGA TTCA -3’; lower cases including a BamHI linker sequence) and Tol2TP-R (5’– gc tctaga ctcgag CTACT CAAAG TTGTA AAACC TCA –3’; lower cases including an XbaI and XhoI linker sequence). The amplified fragment was digested with BamHI and XbaI, and subcloned into the BamHI and XbaI sites of pCS107X, in which the multicloning site of pCS107 was replaced with a new one (5’- ggatcc atcgat ttcgaa gaattc agatct at gcggccgc at aggcct accggt acgcgt ctcgag tctaga -3’; made by Asuka Takahashi and M. T.) to construct pCS107X-Tol2TP. Similarly, the Tol2TP coding region was inserted into the BamHI and XhoI sites of pGEX-6P-1 to make pGEX-6P-Tol2TP. Plasmid constructs were verified by DNA sequencing using an ABI PRISM 3100 Genetic Analyzer. Escherichia coli BL21(DE3) cells carrying the plasmid pT-Trx or pT-GroE for the expression of thioredoxin or chaperon protein GroE, respectively [10] were transformed with pGEX-6P-Tol2TP for producing GST-Tol2TP fusion protein. Expression and purification of GST-Tol2TP E. coli cells carrying pGEX-6P-Tol2TP and pT-Trx or pT-GroE were cultured overnight at 37 ˚C in the LB medium containing 100 g/ml ampicillin and 30 g/ml chloramphenicol, transferred to pre-warmed LB medium/ampicillin/ chloramphenicol at 1:100 dilution, and incubated at 18˚C with shaking at 250 rpm. When OD600 of bacterial culture had reached 0.4-0.6, the expression of GST-Tol2TP was induced with 0.1 mM isopropyl--d-thiogalactopyranoside (IPTG) for 4 h. The cells were collected by centrifugation and frozen in liquid nitrogen until use. All subsequent steps were performed at 0-4˚C. Frozen cells were resuspended in pre-cooled sonication buffer (150 mM NaC1, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM DTT) containing 0.5mM PMSF, and lysed by sonication. The lysate was supplemented with TritonX-100 and PMSF to make final concentrations of 1% and 1 mM, respectively, and shaken gently for 30 min and centrifuged at 18,000 x g for 45 min. The supernatant was loaded onto a glutathione-Sepharose 4B column (GE Healthcare Life Science) pre-equilibrated with sonication buffer containing 1% TritonX-100. After washing with sonication buffer, GST-Tol2TP was eluted with sonication buffer containing 10 mM reduced glutathione. Cleavage and purification of rTol2TP from GST-Tol2TP The eluate containing GST-Tol2TP was added with PreScission protease (8 U for 5–10 mg of fusion protein; GE Healthcare Life Science) and dialyzed with PreScission cleavage buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1 mM DTT, 0.01% TritonX-100) at 4 ˚C for 12–16 hr to cleave the Tol2TP portion (rTol2TP) from GST-Tol2TP. The dialysate was loaded on a glutathione-Sepharose 4B column to obtain rTol2TP as a flow-through fraction. The flow-through fraction was dialyzed with storage buffer (20 mM HEPES, pH 7.4, 500 mM NaCl), concentrated with VIVASPIN (Sartorius), added with glycerol to make a final concentration of 10%, and stored at –80 ˚C until use. The protein concentration of rTol2TP was determined by SDS-polyacrylamide gel electrophoresis (PAGE) and an image analyzer (Odyssey; LI-COR) after staining with Coomassie Brilliant Blue R250 (CBB) using bovine serum albumin (BSA) as a standard. Western blot was performed as described [16] with rabbit anti-GST IgG antibody (Sigma) and anti-rabbit IgG (H+L) antibody conjugated with IRDye800 (Rockland). Reference (Supplement) [16] Shibata, M., Itoh, M., Hikasa, H., Taira, S. and Taira, M. (2005) Role of Crescent in convergent extension movements by modulating Wnt signaling in early Xenopus embryogenesis. Mech. Dev. 122, 1322-1339. [17] Kaufman, P.D. and Rio, D.C. (1992) P element transposition in vitro proceeds by a cut-and-paste mechanism and uses GTP as a cofactor. Cell 69, 27-39. [18] Zhou, L., Mitra, R., Atkinson, P.W., Hickman, A.B., Dyda, F. and Craig, N.L. (2004) Transposition of hAT elements links transposable elements and V(D)J recombination. Nature 432, 995-1001. [19] Rommens, C.M., van Haaren, M.J., Nijkamp, H.J. and Hille, J. (1993) Differential repair of excision gaps generated by transposable elements of the 'Ac family'. Bioessays 15, 507-12. [20] Kawakami, K., Koga, A., Hori, H. and Shima, A. (1998) Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17-22. [21] Kawakami, K. and Shima, A. (1999) Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239-44. [22] Ma, Y. et al. (2004) A biochemically defined system for mammalian nonhomologous DNA end joining. Mol Cell 16, 701-13. Supplement figures Purification of rTol2TP Pilot experiments to determine the optimal culture conditions were performed as follows. To suppress the formation of insoluble aggregates, GST–Tol2TP was coexpressed with thioredoxin (Trx) or GroE in E. coli, and the E. coli cells were cultured at 18 °C. As shown in Supplement Fig. 1, IPTG induced a 100-kDa protein, which is consistent with the calculated molecular mass of GST–Tol2TP (100,017 Da), in the presence of GroE but not Trx. This band was confirmed as GST–Tol2TP on a western blot probed with anti-GST antibody (see Fig. 1B). To make a large preparation of rTol2TP, E. coli carrying pGEX–6P–Tol2TP and pT–GroE were grown in a 2 L culture, and induced with IPTG. SDS–PAGE of the cell lysate showed that a certain amount of GST–Tol2TP was present in the supernatant, although a larger amount of GST–Tol2TP occurred in the pellet (Supplement Fig. 1, lanes 3, 4). The GST–Tol2TP in the supernatant was purified with a glutathione–Sepharose 4B column. A model of Tol2TP action The Tol2 element is flanked by an 8-bp direct repeat unit, TCAAGAAC, resulting from target site duplications during its integration. After the excision of the Tol2 element, this direct repeat is cancelled to restore the original sequence, possibly via a ‘cut-and-paste’ mechanism (Fig. 2D, type I) [17] (Supplement Fig. 2A). However, additional short sequences were present in the footprint sequences of the plasmid DNA after excision. These additional sequences can be categorized as two types. Type II has short direct repeats, as exemplified by clone 2b, as well as by clones 9 and 10c of a previous report [8], whereas type III has a complete or partial sequence complementary to the 8-bp direct repeat unit (GTTCTTGA, GTT, or GA), together with the short direct repeat, as exemplified by clones 5c, 4a, and 2c, as well as by clones 2, 4, 6, 7, 10a, and 10b of a previous report [8]. Hermes transposase excises the element via double-stranded breaks and forms hairpin structures at the ends of the DNA. This activity requires the DDE catalytic triad in the three-dimensional structure of the transposase [18]. Because the DDE catalytic triad is also conserved in Tol2TP (see Fig. 1A), it is likely that the footprint sequences generated by Tol2TP are created through hairpin formation in broken DNA. If this is the case, the type II and type III footprints of rTol2TP can be explained with the model previously proposed by Coen [19] (Fig. 2D, Supplement Fig. 2D). It should be noted that the proportions of type I, II, and III footprint sequences seem to vary greatly between zebrafish [20,21] and Xenopus [[8] and this work]. This implies that Tol2TP may only excise the Tol2 element from DNA, and that the resultant double-stranded breaks of the plasmid DNA are joined by cellular repair enzymes, which could differ in activity between zebrafish and Xenopus embryos. Figure legend (Supplement) Supplement Fig. 1. Purification of GST-Tol2TP Cell cultures with (+) or without (-) IPTG (0.1 mM)-induction and glutathione affinity column purification steps as indicated were analyzed by SDS-PAGE. Dot, GST-Tol2TP band; f.t., flow-through fraction; thick arrow, GST-Tol2TP. Supplement Fig. 2. Models for the transposition of the Tol2 element (A) Precise excision of the Tol2 element results in a single direct repeat unit. (B) the “Coen” model modified by Roomens et al [19]. According to this model, DNA double stranded breaks with 1 bp staggered cuts are generated and formed hairpin structures at the position adjacent to the transposable element. These hairpin structures are resolved by nicks and both open ends of genomic DNA are ligated to create various inversions or direct repeats which depend on the position of nicks. White and black triangles, direct repeat unit and its complementary strand, respectively. (C) A model for the formation of footprint type III. Arrow, positions of nicks for opening hairpin structures; italic, complement sequence. Nonhomologous end joining (NHEJ) of DBS is likely to be executed by a repair enzyme complex containing DNA-dependent protein kinase, Artemis endonuclease, and XRCC4:DNA ligase IV [22]. This model is supported by the observation that, 7 bases, TCAAGAA (left) or CAAGAAC (right), but not 8 bases of the direct repeat unit were connected to the complementary sequence as shown in clones 5c and 4a (Fig. 2D)