Detection of de Novo Insertion of the Medaka Fish Transposable Element Tol2

Corresponding author: Hiroshi Hori, Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan., hori{at}bio.nagoya-u.ac.jp (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Tol2 is a terminal-inverted-repeat transposable element of the medaka fish Oryzias latipes. It is a member of the hAT (hobo/Activator/Tam3) transposable element family that is distributed in a wide range of organisms. We here document direct evidence for de novo insertion of this element. A Tol2 clone marked with the bacterial tetracycline-resistance gene was microinjected into fertilized eggs together with a target plasmid, and the plasmid was recovered from embryos. The screening of plasmid molecules after transformation into Escherichia coli demonstrated transposition of tet into the plasmid and, by inference, precise insertion of Tol2 in medaka fish cells. De novo excision of Tol2 has previously been demonstrated. The present study provides direct evidence that the Tol2 element has the entire activity necessary for cut-and-paste transposition. Some elements of the mariner/Tc1 family, another widespread group, have already been applied to development of gene tagging systems in vertebrates. The Tol2 element of the hAT family, having different features from mariner/Tc1 family elements, also has potential as an alternative gene tagging tool in vertebrates.

SEVERAL transposable elements of the terminal-inverted-repeat class are known to exist in vertebrate genomes. However, only a few of them have been demonstrated to be active. The first example for which a transposition event was observed is the Tzf element of zebrafish (LAM et al. 1996 ). Other examples are the synthetic element Sleeping Beauty (IVICS et al. 1997 ) and exogenous elements such as Tc3 of Caenorhabditis elegans (RAZ et al. 1997 ) and mariner of Drosophila (FADOOL et al. 1998 ). All these elements are members of the mariner/Tc1 transposable element family. There is one other element for which part of the transposition reaction has been demonstrated. The element is Tol2, found in the medaka fish (KOGA et al. 1996 ), and de novo excision has also been detected in zebrafish (KAWAKAMI et al. 1998 ). However, de novo insertion has hitherto not been observed. This element is a member of the hoto/Activator/Tam3 (hAT) element family found in various organisms, even across kingdoms (CALVI et al. 1991 ; ATKINSON et al. 1993 ; KOGA et al. 2000 ). Such widespread occurrence is an important feature for developing a gene tagging system because it implies relative independence of transposition mechanisms from species-specific host factors and, thus, applicability to a relatively wide range of organisms.

The purpose of this study was to detect de novo insertion of Tol2. A putative transposase of Tol2 has already been suggested (KOGA et al. 1999 ) and catalysis of excision has been demonstrated (KAWAKAMI and SHIMA 1999 ). The same enzyme would also be expected to catalyze insertion, as is usually the case with terminal-inverted-repeat elements. The reason de novo insertion has not been detected earlier is that it requires, in contrast to the excision case, a suitable experimental system to allow detection of the inserted element at what would normally be random locations. One strategy to overcome this difficulty is to trace a genetically marked element. In this study, we marked Tol2 with the bacterial tetracycline-resistance (Tet^R) gene and screened target DNA fragments in Escherichia coli transformants for transposition products conferring tetracycline resistance. Precise insertion, with Tol2 flanked by 8-bp target site duplications, was detected. In addition, the transposition was proven to be catalyzed by the transposase that is encoded by a sequence within Tol2.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fish:
Oryzias latipes (medaka fish, also called Japanese medaka fish) and O. melastigma (Indian medaka fish) were used. O. latipes was purchased from a pet shop in Nagoya and the fish contained 20 Tol2 copies per diploid genome according to genomic Southern blots (KOGA and HORI 1999 ). O. melastigma was obtained from the World Medaka Aquarium of the Nagoya City Higashiyama Zoological Garden. This species does not harbor Tol2 in its genome (KOGA et al. 2000 ).

Construction of plasmids:
Tol2 was first identified as a 4.7-kb insertion sequence in the tyrosinase gene of an albino mutant fish (KOGA et al. 1996 ). This particular Tol2 copy (Tol2-tyr, GenBank accession no. D84375) together with its 8-bp target site duplication sequences was amplified by PCR. The primers used were 5'-AAGGATCCTCAAGAACCAGAGGTGTAAAGT-3' and 5'-CCTCTAGAGTTCTTGACAGAGG TGTAAAAA-3'. The third to eighth nucleotides are restriction enzyme sites (BamHI and XbaI, respectively) for cloning, the next eight nucleotides are target site duplications (part of the tyrosinase gene), and the remaining sequences to 3' are the ends of Tol2 (the left and right ends, respectively). The amplified fragment was cloned into pHSG399 (TAKESHITA et al. 1987 ) at its BamHI and XbaI sites in the lacZ gene. An internal region of Tol2 between its two PstI sites (positions 2401 and 4272 on the Tol2-tyr sequence) was removed and replaced with the Tet^R gene of pBR322 amplified with PCR (nucleotides 4227–1375, including nucleotide 1, of accession no. J01749). This Tol2-carrying donor plasmid was denoted pDon01 (Fig 1). The target plasmid pTar01 (Fig 1) was prepared by inserting a 0.8-kb DNA fragment (nucleotides 2679–747, including nucleotide 1, of accession no. M12787 for pHSG664; HASHIMOTO-GOTOH et al. 1986 ) into pHSG299 (TAKESHITA et al. 1987 ). The fragment contains the streptomycin-sensitive (Str^S) gene whose disruption, in association with a specific bacterial strain, confers the streptomycin-resistance phenotype. It was prepared with the long-range plan of screening for insertions in this gene. In the present study, the 0.8-kb fragment was employed simply to enlarge the target plasmid.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Organization of plasmids. pDon01 is a donor plasmid. It contains the chloramphenicol-resistance (CamR) gene and a Tol2 element carrying the tetracycline-resistance (TetR) gene. pTar01 is a target plasmid containing the kanamycin-resistance (KanR) gene.

Transposition assay:
The scheme of the transposition assay is illustrated in Fig 2. The pDon01 and pTar01 plasmids, 150 ng/µl each in 10 mM Tris-HCl (pH 8.0), were microinjected into fertilized eggs at the one-cell stage, with or without mRNA for the putative Tol2 transposase (KOGA et al. 1999 ), prepared by in vitro transcription using the RiboMAX large scale RNA production system SP6 (Promega, Madison, WI). A mammalian-cap-like sequence was added to its 5' end and the mRNA was included in the DNA solution for injection at a final concentration of 150 ng/µl. After incubation at 25° for 24 hr, plasmid molecules were recovered from embryos by the method of HIRT 1967 and then introduced into the JM109 strain of E. coli by electroporation. The bacteria were screened for plasmids showing both the Kan^R and Tet^R phenotypes, which can be expected to include products of transposition. The drug concentrations were 50 µg/ml for kanamycin and 6 µg/ml for tetracycline.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Transposition assay using plasmids. Plasmid molecules recovered from embryos were introduced into bacteria by electroporation. Most of the bacteria were plated on kanamycin- and tetracycline-containing media to screen for Tol2 insertion into the pTar01 plasmid. Small aliquots were plated on media containing only kanamycin to estimate the number of pTar01 molecules both with and without Tol2 insertion.

Sequencing analysis:
Candidate transposition products were further selected by restriction mapping and sequenced for their Tol2 terminal and flanking regions, as described (KOGA et al. 1995 ). The sequencing primers were nucleotides 424–401 of the Tol2-tyr sequence for the left terminus and nucleotides 4316–4339 for the right terminus.

	RESULTS

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Transposition assay:
We carried out five experiments as listed in Table 1. In experiments A–D, which included microinjection of plasmid DNAs, plasmids showing both the Kan^R and Tet^R phenotypes were found. Twenty plasmids each from the four experiments were randomly chosen and checked for their sizes by agarose gel electrophoresis (Fig 3). This test revealed the majority of the plasmids to be of two sizes: 7.7 and 10 kb. Plasmids of 7.7 kb were observed only in experiments B and D, in which mRNA was microinjected together with the plasmid DNAs. If the Tol2 portion (4274 bp) of pDon01 (6517 bp) is integrated precisely into pTar01 (3446 bp), the expected size of a resultant plasmid is 7.7 kb. Therefore, these plasmids are likely to be products of precise Tol2 insertion. The fact that all these plasmids were found to carry single SacI cutting sites also supports this inference because pDon01 and pTar01 both have single SacI sites in their vector portions (see Fig 1).

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Electrophoresis of plasmid clones. Single plasmids isolated from experiments A–D were digested with SacI and electrophoresed on 0.6% agarose gels. In the example shown from experiment B, three clones (B-1, -5, and -7) exhibit single fragments of 7.7 kb, six clones (B-2, -3, -4, -6, -9, and -10) have a total length of 10 kb, and one clone (B-8) is >12 kb.

If the pDon01 and pTar01 plasmids were combined into one molecule, the resultant plasmid would be expected to be 10 kb in size, like the plasmids of this size observed in experiments A–D. They all demonstrated two SacI sites (examples shown in Fig 3) consistent with the inferred link. The fact that restriction patterns for SacI differed among these 10-kb plasmids suggested that the integrations, on the assumption of this inference, were independent events.

There were also a few plasmids sized >12 kb with three SacI sites, as shown in Fig 3.

Sequencing analysis:
Of the 7.7-kb plasmids, five each from experiments B and D were randomly chosen and sequenced for their Tol2 terminal and flanking regions. With all plasmids and for both Tol2 termini, the Tol2 sequences were observed as far as the last nucleotides and were flanked by sequences of pTar01 (Fig 4). In addition, target site duplications of 8 bp were observed in all cases (Fig 4). The insertion breakpoints according to the Tol2-flanking sequences are illustrated in Fig 5. There was no apparent region where insertion breakpoints were concentrated.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 4. DNA sequences of Tol2 ends and their flanking regions. Five clones each from experiments B and D were sequenced. The regions outside Tol2 were all confirmed to be parts of the pTar01 plasmid. Target site duplications of 8 bp, in boldface type, were observed in all the cases.

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Locations of Tol2 insertions on the pTar01 plasmid. The stippled arrows indicate individual Tol2 copies, in accordance with the direction of the transposase gene in the element. Clones from experiment B are shown above and those from experiment D below the plasmid.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Detection of de novo insertions:
Our results provide evidence for de novo insertion of Tol2. Because excision has already been detected, it can now be concluded that Tol2 has all the activity necessary for cut-and-paste transposition.

We have not conducted further analysis of the 10-kb plasmids and the few larger plasmids because the purpose of our study was to detect insertion of Tol2, attained with the 7.7-kb plasmids. The 10-kb plasmids appear to have resulted from simple combination of pDon01 and pTar01 into one plasmid. If this is true, a possible mechanism for this event is homologous recombination, because the two vector plasmids (pHSG399 and pHSG299) share regions inherited from their common origin. Irrespective of the mechanisms involved, the recombination events occurred not in bacteria but in medaka fish cells because Kan^R Tet^R plasmids were not observed in experiment E. Precise insertion reaction leading to 7.7-kb plasmids also occurred in medaka fish cells.

Evidence for transposase function:
O. melastigma does not contain Tol2 in its genome. Therefore, the difference in results between experiments C and D implies that the mRNA is essential for transposition of Tol2. It is thus evident that the mRNA encodes a transposase of Tol2 that also functions as such in O. melastigma.

O. latipes contains 20 Tol2 copies and endogenous mRNA molecules are present in cells of this species (KOGA et al. 1999 ). We infer from the results of experiments A and B that the amount is relatively low and the supply of exogenous mRNA by microinjection was responsible for invoking transposition of Tol2.

Development of a monitoring system:
Our detection system, if applied on a larger scale, would be useful for determining transposition frequency and, thus, finding factors that affect the transposition. However, there is a problem that remains to be solved and that is the presence of 10-kb plasmids. They can be eliminated by restriction enzyme digestion and agarose gel electrophoresis but a more efficient device is desirable. One possible approach is to use the Str^S gene already contained in the pTar01 plasmid (see MATERIALS AND METHODS). Screening with streptomycin, in addition to kanamycin and tetracycline, would be useful for collecting only insertions in the Str^S gene.

Potential as a gene tagging tool:
Some elements of the mariner/Tc1 family have already been applied for developing a gene tagging system in vertebrates. However, hAT family elements have different features from those of the mariner/Tc1 family, for example, being larger in size. This might be an advantage for carrying large DNA fragments. One element, Activator of maize, has been shown to transpose preferentially within the same chromosome (GREENBLATT 1984 ; DOWE et al. 1990 ; MORENO et al. 1992 ) and, moreover, in a systematic further analysis, preferentially to nearby regions (MACHIDA et al. 1997 ). Such a transposition would be beneficial to target nearby genes or regions. This might lead to creation of chromosomal deletions and inversions and to generating large numbers of mutations within a single gene. Thus, the Tol2 element of the hAT family can be expected to provide a gene tagging tool for molecular techniques as alternatives to those using mariner/Tc1 family elements.

	ACKNOWLEDGMENTS

We are grateful to H. Hashikawa, M. Sato, and S. Susaki for providing the O. melastigma samples. We also thank H. Ohtsubo and Y. Sekine for helpful discussions. This work was partly supported by grant no. 10216025 to A.K. and no. 09554053 to H.H. from the Ministry of Education, Science, Sports, and Culture of Japan, and also by the Takeda Science Foundation to A.K.

Manuscript received April 24, 2000; Accepted for publication July 10, 2000.

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