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. 2006 Oct;174(2):639-49.
doi: 10.1534/genetics.106.060244. Epub 2006 Sep 7.

Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition

Akihiro Urasaki  1 , Ghislaine Morvan, Koichi Kawakami
Affiliations

Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition

Akihiro Urasaki et al. Genetics. 2006 Oct.

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 (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 >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, 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 for understanding roles of the repetitive sequence in the subterminal region in transposition.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
The structures and activities of mini-Tol2 constructs. (A) The structures of the full-length Tol2, T2KXIGΔin, and mini-Tol2 constructs created in this study and their activities in the excision assay. The full-length Tol2 (4682 bp) encodes the transposase gene. Exons are shown by gray (untranslated region) and black (translated region) boxes. Lines (exons), dotted lines (intron), and AAAA (polyadenylation) above Tol2 indicate mRNA for the transposase. The left end (5′-end) and the right end (3′-end) are designated with respect to orientation of the transcript. The mini-Tol2 constructs contain various lengths of DNA from the left-end (purple) and the right-end (blue) sequences. All of these constructs contain the GFP expression cassette in the middle. The excision frequency represents “the number of DNA samples from which the excision product was amplified” per “the number of DNA samples analyzed.” (B) The examples of the excision assay performed in this study. DNA samples prepared from embryos injected with the transposase mRNA and plasmids containing T2AL200R200G, T2AL175R200G, T2AL150R200G, T2AL200R150G, and T2AL200R100G were analyzed by PCR. Top bands represent PCR products from unexcised transposon constructs and bottom bands represent excision products. Lanes 1–13: PCR products amplified from DNA samples prepared from each injected embryo. Lane 14: positive control (a DNA sample was prepared from an embryo injected with T2KXIGΔin). Lane 15: negative control (no transposase mRNA). Lane 16: negative control (a DNA sample was prepared from an uninjected embryo). The weak bands are seen in T2AL175R200G (lanes 10, 11, 15) and T2AL200R100G (lanes 1–13, 15) artifacts, which tend to be amplified when the amount of the excision product is too low to be amplified.
F<sc>igure</sc> 2.—
Figure 2.—
Germline transmission using mini-Tol2 constructs. (A, D, F) GFP expression in transgenic embryos carrying a single insertion of the mini-Tol2 constructs T2KXIGΔin (A), T2AL200R200G (D), and T2AL200R150G (F). (B, C, E, F, H, I) Southern blot hybridization analysis of transposon insertions in F1 fish. F1 fish from founder fish injected with the transposase mRNA and a plasmid containing T2KXIGΔin (B and C), T2AL200R200G (E and F), or T2AL200R150G (H and I) were analyzed by Southern blot hybridization. The genomic DNA was prepared from tail fins of 12 F1 transgenic fish from each founder fish (T2KXIGΔin-4, T2KXIGΔin-5, T2AL200R200G-2, T2AL200R200G-9, T2AL200R150G-1, and T2AL200R150G-2: see Table 1) and were used for the Southern blot analysis.
F<sc>igure</sc> 3.—
Figure 3.—
Spacing capacity and requirements. (A) The structures of transposon constructs carrying various lengths of inserts between the left and right ends, their total lengths, and activities determined by germline transmission and the transient excision assay. NcoI sites in T2KIGΔin-9k were used for the Southern blot analysis in B. f1, r1, f2, and r2: positions and directions of primers used for PCR in C. ND, not determined. (B) Southern blot hybridization analysis of NcoI-digested genomic DNA from transgenic fish. The result indicates an insertion of a single copy of T2KIGΔin-9k. (C) The genomic DNA from the transgenic fish carrying the T2KIGΔin-9k insertion (lanes 1 and 3) and the injected plasmid DNA (lanes 2 and 4) were used for PCR. PCR was carried out by using f1 and f2 (lanes 1 and 2) and f2 and r2 (lanes 3 and 4). M, size marker; lane 5, no DNA control. (D) The genomic DNA surrounding the T2KIGΔin-9k integration site was determined by adaptor ligation PCR. The target-site duplication (TSD) is in boldface type. The T2KIGΔin-9k insertion was mapped within an intron of zgc:65966 (Pho GDP dissociation inhibitor).
F<sc>igure</sc> 4.—
Figure 4.—
The left- and right-end sequences of Tol2. The 200-bp left end (5′-end) and the 150-bp right end (3′-end) sequences sufficient and required for transposition. The numbers above the sequence show bases from the left end and the numbers below the sequence show bases from the right end. The 5-bp repeats, 5′-(A/G)AGTA-3′, are in red on the sequence and also indicated by arrows.
F<sc>igure</sc> 5.—
Figure 5.—
The structures and activities of Tol2 constructs with substitution mutations created by the MluI mutagenesis. Boxes at the both ends represent the TIRs and the TIR mutations (mTIRL1-6 and mTIRR1-6) are indicated as solid boxes. Open arrowheads indicate positions and directions of the repeat 5′-(A/G)AGTA-3′, and solid arrowheads indicate repeats mutated by base substitutions shown in Table 4. In mL88-92, mL158-162, and mR89-94, the substitution mutations in the sequences outside of the repeats (Table 4) are shown as solid boxes. Excision frequency represents “the number of DNA samples from which the excision product was amplified” per “the number of DNA samples analyzed.”
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