Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage

Abstract

The Tol2 element of the medaka fish Oryzias latipes belongs to the hAT family of transposons (hobo/Ac/Tam3). We report here identification of a functional transposase of Tol2 that is capable of catalyzing its transposition in the germ line of zebrafish Danio rerio. A transcript produced from Tol2 encodes a putative transposase. Zebrafish fertilized eggs were coinjected with mRNA transcribed in vitro, using cDNA of the Tol2 transcript as a template and a plasmid DNA harboring a mutant Tol2, which had a deletion in the putative transposase gene but retained necessary cis sequences. The injected fish were raised to adulthood and mated to noninjected fish, and genomic DNA of the progeny fish were analyzed by PCR and Southern hybridization. Half of F(1) fish obtained from one of eight injected fish contained the Tol2 DNA in their genomes but not the vector portion. Among these F(1) fish, Tol2 insertions at four different loci were identified, and some F(1) fish carried two or three different Tol2 insertions, indicating that the germ line of the founder fish is highly mosaic. Sequencing analyses revealed that, in all cases, Tol2 was surrounded by zebrafish genomic sequences, and an 8-bp duplication was created at the target site, indicating that Tol2 was integrated in the zebrafish genome through transposition. This study identifies an autonomous member of a DNA-based transposable element from a vertebrate genome. The Tol2 transposon system should thus be used to develop novel transgenesis and insertional mutagenesis methods in zebrafish and possibly in other fishes.

Figure 1

Structures of the Tol2 elements and cDNA. The black and gray boxes indicate the Tol2-tyr and (Tol2-tyr)ΔRV elements, respectively, and thin lines flanking these boxes represent plasmid DNA (the medaka fish tyrosinase sequence). Self-ligation at two EcoRV sites in Tol2-tyr generated (Tol2-tyr)ΔRV. 5′ and 3′ in Tol2 cDNA indicate the direction of transcription, and dotted lines indicate introns. Arrowheads show positions and directions of primers used in the experiment described in Fig. 2A. A thick bar shows the probe used in Southern hybridization analyses. Note that the total length of the Tol2-tyr element, previously reported as 4681 bp (20), has been corrected to 4682 bp (D84375), by addition of 1 bp in the first intron, by the authors of the first report. r9, Tol2r9; f5, Tol2f5; r7, Tol2r7; f20, Tol2f20.

Figure 2

PCR and Southern hybridization analyses of F₁ fish. (A) PCR analyses of genomic DNA extracted from caudal fin clippings of F₁ fish from fkk-1 and fkk-7. Primer pairs used for PCR and the sizes of the PCR products are shown on the right. Two (fkk-1, lanes 1 and 2) and 25 F₁ fish were found positive for PCR using Tol2f5/Tol2r7 out of 68 and 50 F₁ fish from fkk-1 and fkk-7, respectively. PCR analyses of the 25 F₁ fish from fkk-7 yielded the same results, and four representatives are shown (fkk-7, lanes 1–4). The same four samples were used also for Southern hybridization (described in C). In the control (P), 100 ng of zebrafish genomic DNA plus 1 pg of the (Tol2-tyr)ΔRV plasmid DNA were used as a PCR template. (B) Southern hybridization analysis of F₁ fish from fkk-1. DNA samples of two fish (A) were digested with either EcoRV or EcoRI and hybridized with the probe shown in Fig. 1. (C) Southern hybridization analysis of F₁ fish from fkk-7. DNA samples of four fish (A) were digested with either EcoRV or EcoRI and hybridized with the probe (Fig. 1). EcoRI bands of four different sizes, shown as a–d on the right, correspond to the fkk-7a, fkk-7b, fkk-7c, and fkk-7d insertions described in the text. The lower bands in EcoRV lane 1 are probably a triplet. (D) Southern hybridization analysis of F₂ fish from the lane 1 F₁ fish (fkk-7). Ten fish out of 14 F₂ fish, which were positive for PCR using Tol2f5 and Tol2r7, were analyzed. DNA samples were digested with EcoRI and hybridized with the probe (Fig. 1). Fish with fkk-7b alone (lanes 1, 2, 10), fkk-7b alone (lanes 4, 6, 8), and both fkk-7b and fkk-7c (lanes 3, 5, 7, 9) were identified.

Figure 3

Inverse PCR and DNA sequencing analysis of the Tol2 insertions. (A) Primers and restriction enzyme sites used for inverse PCR. The gray box indicates (Tol2-tyr)ΔRV. Arrowheads indicate positions and directions of primers. EcoRV and HindIII are unique sites. Sau3A I and PstI cut more than twice, and only most 3′ sites used to clone the 3′ junctions are drawn. r1, Tol2r1; r9, Tol2r9; f5, Tol2f5; f1, Tol2f1; r2, Tol2r2; r10, Tol2r10; f20, Tolf20; f2, Tol2f. (B) DNA sequences adjacent to (Tol2-tyr)ΔRV. DNA fragments were amplified by inverse PCR from fish with fkk-7a, fkk-7b, fkk-7c, and fkk-7d insertions (Fig. 2C, lanes 1–4), and sequenced. Eight-base pair duplications, created upon transposition, are underlined. (C) Structures of Tol2 insertions. These structures were drawn based on DNA sequences from inverse PCR products and the wild-type DNA fragments. The gray box indicates (Tol2-tyr)ΔRV. Restriction enzyme sites used for inverse PCR are shown. Arrowheads (7a5′ and 7a3′, 7b5′ and 7b3′, 7c5′ and 7c3′, and 7d5′ and 7d3′) indicate positions and directions of primers used to amplify wild-type DNA fragments, which extend across the integration sites for Tol2. 7d5′-2 primer was used in the experiment described in Fig. 4.

Figure 4

Identification of the Tol2 insertions in F₁ fish. (A) Southern hybridization analysis of F₁ fish from fkk-7. DNA samples from 20 F₁ fish were digested with EcoRI and hybridized with the probe (Fig. 1). The numbering of the lanes (fish) is continued from Fig. 2C. Bands of four different sizes, a (lane 24), b (lanes 5–10, 12, 13, 20, 21), c (lanes 5–7, 11, 20, 21), and d (lanes 9–23), were detected. The lower bands on lanes 11, 20, and 21 are doublets. (B) PCR analysis of 24 F₁ fish from fkk-7. DNA samples on lanes 1–4 correspond to those on lanes 1–4 of Fig. 2C. PCR reactions were carried out using the wnt5A primers (positive control) and 7a3′ and Tol2f20 (fkk-7a), 7b5′ and Tol2r1 (fkk-7b), 7c3′ and Tol2f20 (fkk-7c), or 7d5′-2 and Tol2r1 (fkk-7d). On these photos, the presence of the lower band indicates that the fish carries the specific Tol2 insertion. The results are consistent with those obtained by Southern hybridization analysis and are summarized in Table 1.