Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells

The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.     

Targeted reduction of the DNA methylation level with 5-azacytidine promotes excision of the medaka fish Tol2 transposable element

The Tol2 element of the medaka fish Oryzias latipes is a member of the hAT (hobo/Activator/Tam3) transposable element family. There is evidence for rapid expansion in the genome and throughout the species in the past but a high spontaneous transposition rate is not observed with current fish materials, suggesting that the Tol2 element and its host species have already acquired an interactive mechanism to control the transposition frequency. DNA methylation is a possible contributing factor, given its involvement with many other transposable elements. We therefore soaked embryos in 5-azacytidine, a reagent that causes reduction in the DNA methylation level, and examined amounts of PCR products reflecting the somatic excision frequency, obtaining direct evidence that exposure promotes Tol2 excision. Our results thus suggest that methylation of the genome DNA is a factor included in the putative mechanisms of control of transposition of the Tol2 element.     

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans

Tol1 is a DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes, and has been proven to be transposed in various vertebrate species, including mammals. This element belongs to the hAT (hobo/Activator/Tam3) transposable element family, whose members are distributed in a wide range of organisms. It is thus possible that Tol1 is mobile in organisms other than vertebrates. We here show that transposition of this element occurs in the nematode Caenorhabditis elegans. A donor plasmid containing a Tol1 element and a helper plasmid carrying the transposase gene were delivered into gonad cells and, after several generations of culturing, were recovered from worms. PCR analysis of the donor plasmid, using primers that encompassed the Tol1 element, revealed excision of the Tol1 portion from the plasmid. Analysis of genomic DNA of the worms by the inverse PCR method provided evidence that Tol1 had been integrated into the C. elegans chromosomes. Vertebrates and C. elegans are phylogenetically distantly related organisms in that the former are deuterostomes and the latter a protostome animal. Our results indicate (1) the transposition reaction of the Tol1 element requires, besides the transposase, no factors from host cells, or (2) the host factors, even if required, are those that are common to protostomes and deuterostomes. The results also have significance for the development of a gene transfer vector and other biotechnology tools for C. elegans.     

Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis

The Tol2 transposable element from the medaka fish belong to the hAT family of transposons. In the previous studies, we have identified an autonomous member of this element, which encodes a fully functional transposase, and have shown that it can catalyze transposition in the zebrafish germ lineage. To date, the Tol2 element is the only natural transposon in vertebrates from which an autonomous member has been identified. We report here transposase-dependent excision of the Tol2 element in Xenopus laevis and Xenopus (Silurana) tropicalis embryos. We coinjected a plasmid DNA containing a nonautonomous Tol2 element and the transposase mRNA synthesized in vitro into two-cell-stage embryos, and analyzed DNA extracted from the injected embryos by polymerase chain reaction (PCR). We demonstrated that the Tol2 element could be excised from the plasmid DNA in both X. laevis and X. tropicalis only when it was coinjected with the transposase mRNA. In most cases, a complete loss of the Tol2 sequence was accompanied by addition of a short DNA sequence to the target sequence, indicating that transposase-dependent excision occurred. While these footprints were characteristic to those created upon excision of transposons of the hAT family, the additional bases found in Xenopus were longer and their structures were more complicated than those detected upon excision in zebrafish. This may reflect differences in the activities of host factors involved in either transposition, repair, or both between fish and frog. Our present study suggests that the Tol2 transposon system should be used as a novel genetic tool to develop transgenesis and mutagenesis methods in Xenopus.     

PCR detection of excision suggests mobility of the medaka fish Tol1 transposable element in the frog Xenopus laevis

Tol1 is a DNA-based transposable element identified in the medaka fish Oryzias latipes and a member of the hAT (hobo/Activator/Tam3) transposable element family. Its mobility has already been demonstrated in the human and mouse, in addition to its original host species. This element is thus expected to be useful in a wide range of vertebrates as a genomic manipulation tool. Herein, we show that the Tol1 element can undergo excision in the African clawed frog Xenopus laevis, a major model organism for vertebrate genetics and developmental biology. An indicator plasmid carrying a Tol1 element was injected into 2- or 4-cell-stage embryos together with either a helper plasmid coding for the full-length Tol1 transposase or a modified helper plasmid yielding a truncated protein, and recovered from tailbud-stage embryos. Deletion of the Tol1 region of the indicator plasmid was observed in the experiment with the full-length transposase, and not in the other case. The deletion was associated with various footprint sequences at breakpoints, as frequently observed with many DNA-based transposable elements. These results indicate that the Tol1 element was excised from the indicator plasmid by catalysis of the transposase, and suggest that the Tol1 element is mobile in this frog species.     

Gene Transfer and Cloning of Flanking Chromosomal Regions Using the Medaka Fish Tol2 Transposable Element

For the ultimate purpose of developing genetic tools using the medaka fish Tol2 transposable element, we examined whether it can transfer a marker gene into the fish genome and also be applied for cloning of chromosomal regions adjacent to insertion points. An internal region of Tol2 was removed and replaced with the green fluorescent protein (GFP) gene and a bacterial plasmid replication origin. This modified Tol2 clone was microinjected into fertilized eggs together with messenger RNA for the Tol2 transposase. The GFP gene was found to be integrated into chromosomes and transmitted to subsequent generations. Restriction enzyme digestion of genomic DNA of a transformant fish, followed by ligation and introduction into bacteria, produced a plasmid containing the entire element and flanking chromosomal regions. Sequencing analysis of this clone demonstrated transposition of the element in the germline of the first generation. Thus, the basic requirements for a gene transfer vector and gene tagging system were fulfilled. Received July 30, 2001; accepted October 4, 2001     

Transposition of the vertebrate Tol2 transposable element in Drosophila melanogaster

The Tol2 element is a transposon found from a genome of a vertebrate, a small teleost medaka fish. Tol2 encodes a gene for a transposase which is active in vertebrate animals so far tested; for instance, in fish, frog, chicken and mammals, and transgenesis methods using Tol2 have been developed in these model vertebrates. However, it has not been known whether Tol2 can transpose in animals other than vertebrates. Here we report transposition of Tol2 in an invertebrate Drosophila melanogaster. First, we injected a transposon donor plasmid containing a Tol2 construct and mRNA encoding the Tol2 transposase into Drosophila eggs, and found that the Tol2 construct could be excised from the plasmid. Second, we crossed the injected flies, raised the offspring, and found that the Tol2 construct was integrated into the genome of germ cells and transmitted to the next generation. Finally, we constructed a Tol2 construct containing the white gene and injected the transposon donor plasmid and the transposase mRNA into fertilized eggs from the white mutant. We analyzed their offspring, and found that G1 flies with wild type red eyes could be obtained from 35% of the injected fly. We cloned and sequenced 34 integration loci from these lines and showed that these insertions were indeed created through transposition and distributed throughout the genome. Our present study demonstrates that the medaka fish Tol2 transposable element does not require vertebrate-specific host factors for its transposition, and also provides a possibility that Tol2 may be used as a new genetic tool for transgenesis and genome analysis in Drosophila.     

Does the proposed DSE motif form the active center in the Hermes transposase?

Donor cleavage and strand transfer are two functions performed by transposases during transposition of class II transposable elements. Within transposable elements, the only active center described, to date, facilitating both functions, is the so-called DDE motif. A second motif, R-K-H/K-R-H/W-Y, is found in the site-specific recombinases of the tyrosine recombinase family. While present in many bacterial insertion sequences as well as in the eukaryotic family of mariner/Tc1 elements, the DDE motif was considered absent in other classes of eukaryotic class II elements such as P, and hAT and piggyBac. Based on sequence alignments of a hobo-like element from the nematode Caenorhabditis elegans, to a variety of other hAT transposases and several members of the mariner/Tc1 group, Bigot et al. [Gene 174 (1996) 265] proposed the presence of a DSE motif in hAT transposases. In the present study we tested if each of these three residues is required for transposition of the Hermes element, a member of the hAT family commonly used for insect transformation. While D402N and E572Q mutations lead to knock-out of Hermes function, mutations S535A and S535D did not affect transposition frequency or the choice of integration sites. These data give the first experimental support that D402 and E572 are indeed required for transposition of Hermes. Furthermore, this study indicates that the active center of the Hermes transposase differs from the proposed DSE motif. It remains to be shown if other residues also form the active site of this transposase.     

Low transposition frequency of the medaka fish Tol2 element may be due to extranuclear localization of its transposase

Transposase proteins of some highly active DNA-based transposable elements, such as the maize Activator element, are known to possess nuclear localization signals (NLSs). We examined if this is also the case for the transposase of the medaka fish Tol2 element, a member of the hAT (hobo/Activator/Tam3) transposable element family, using human and mouse culture cells. Unexpectedly, the transposase-lacZ fusion protein, in which the lacZ is a location marker, was found to be present in the cytoplasm rather than in the nucleus, suggesting that the Tol2 transposase contains a signal for extranuclear localization. The same staining pattern was also observed with a fusion protein containing a 33-amino-acid region at about the center of the primary structure of the transposase. The Tol2 element might have a mechanism to control its transposition frequency that includes extranuclear localization of its transposase.     

Long and short mRnas transcribed from the medaka fish transposon Tol2 respectively exert positive and negative effects on excision

The medaka fish transposable element, Tol2, is a member of the hAT family of transposons. It has been directly demonstrated to be active and two mRNAs, differing in length, have been isolated. They cover exons 1-4 and exons 2-4 and the longer form has already been proven to catalyse transposition reactions. However, the function of the shorter mRNA in medaka cells has hitherto remained unclear. In the present study, first we constructed a quantitative system to detect Tol2 excision using an indicator plasmid carrying a non-autonomous Tol2 within its lacZ gene; second we injected mRNAs with the plasmid into medaka eggs. Excision of Tol2 was detected as E. coli blue colonies caused by the recovery of lacZ activity. Addition of the longer mRNA increased excision, but the shorter did not. Moreover, co-injection of both mRNAs greatly lowered the frequency compared with the case of treatment with the longer mRNA alone. These results indicate that the shorter mRNA has an inhibitory effect on the excision reaction, and that the N-terminal region of the transposase encoded by exon 1, including a BED zinc finger, presumably plays an important role in excision. Here, we suggest a regulatory mechanism of Tol2 transposition involving the expression of these mRNAs.     

The Tol2 transposable element of the medaka fish: an active DNA-based element naturally occurring in a vertebrate genome

Several DNA-based transposable elements are known to be present in vertebrate genomes, but few of them have been demonstrated to be active. The Tol2 element of the medaka fish is one such element and, therefore, is potentially useful for developing a gene tagging system and other molecular biological tools applicable to vertebrates. Towards this goal, analyses of the element at the molecular, cellular and population levels are in progress. Results so far obtained are described here.     

Homogeneity in the structure of the medaka fish transposable element Tol2

The hAT family is a group of transposable elements of the terminal inverted repeat class, which includes Ac of maize, hobo of Drosophila and Tam3 of Antirrhinum (snapdragon). All the members of this family so far examined are known to comprise complete and defective copies, with a good correspondence to autonomous and non-autonomous elements, respectively. Internal deletion is the most common cause of defective copies. Tol2, a transposable element of the medaka fish Oryzias latipes, is a member of the hAT family. We examined, mainly by the genomic Southern blot analysis, variation in the structure of copies of this element, and revealed that there are few or no internally deleted copies. This situation is unusual in a member of the hAT family. Possible causes of this anomaly are discussed.     

Tol1转座子研究进展

Tol1和Tol2是在青鳉基因组中发现的具有自主活性的DNA转座子,而Tol1转座子的自主活性是新近才发现的,因此对它的报道较少。较之Tol2,Tol1可以携带更大片段的DNA进行转座,且Tol1的转座不受转座酶"过量表达抑制"的影响。研究已证实,Tol1转座子在秀丽线虫、斑马鱼、爪蟾和人等多种生物中具有转座活性。因此,在动物转基因和基因功能研究等方面有重要的应用前景。从Tol1转座子的结构特征、转座机制和作为基因转移载体的优点,以及应用研究等方面进行了简要的综述。     

金鱼hAT家族转座子Tgf2的克隆及其结构

hAT家族转座子以果蝇hobo、玉米Ac和金鱼草(Ceratophyllum demersum L.)Tam3为代表,以"剪切-粘帖"方式进行DNA转座。1996年,日本学者首次在白化青鳉(Oryzias latipes)中发现具有天然活性的脊椎动物hAT家族转座子,即青鳉Tol2转座子,该转座子已在模式生物斑马鱼转基因、基因和启动子捕获方面进行了广泛应用。文章根据玉米Ac与青鳉Tol2转座子序列保守区设计一对引物,在19种不同鱼类物种或品系中进行PCR筛选,最后发现此类hAT家族转座子在我国不同品系金鱼中存在,命名为金鱼Tgf2转座子。金鱼Tgf2转座子全长4720bp,由4个阅读框组成,与青鳉Tol2转座子的相似度为97%。金鱼Tgf2与青鳉Tol2转座子在末端倒位重复和亚末端重复上存在一定差异,此外,金鱼Tgf2转座子的中间反向重复序列(1453bp到2091bp)可形成一种"十"字结构,明显有别于青鳉Tol2转座子形成的茎环结构,这些区域与转座活性密切相关。文章预示金鱼Tgf2转座子可能具有更高的天然转座活性,构建高效金鱼Tgf2转基因元件可供鱼类转基因和基因捕获研究。     

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.     

A rice Tc1/mariner-like element transposes in yeast

The Tc1/mariner transposable element superfamily is widely distributed in animal and plant genomes. However, no active plant element has been previously identified. Nearly identical copies of a rice (Oryza sativa) Tc1/mariner element called Osmar5 in the genome suggested potential activity. Previous studies revealed that Osmar5 encoded a protein that bound specifically to its own ends. In this report, we show that Osmar5 is an active transposable element by demonstrating that expression of its coding sequence in yeast promotes the excision of a nonautonomous Osmar5 element located in a reporter construct. Element excision produces transposon footprints, whereas element reinsertion occurs at TA dinucleotides that were either tightly linked or unlinked to the excision site. Several site-directed mutations in the transposase abolished activity, whereas mutations in the transposase binding site prevented transposition of the nonautonomous element from the reporter construct. This report of an active plant Tc1/mariner in yeast will provide a foundation for future comparative analyses of animal and plant elements in addition to making a new wide host range transposable element available for plant gene tagging.     

Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements

Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposition is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic distribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydrogen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements.     

Reversion mutation of ib oculocutaneous albinism to wild-type pigmentation in medaka fish

We have previously identified three naturally occurring mutations in the medaka fish tyrosinase gene caused by transposable element insertions. Tyr-i(b) is one of these, containing the Tol2 element in the promoter region. Its homozygous carriers exhibit a weak oculocutaneous albino phenotype. We report here spontaneous reversion of the albino phenotype to the wild-type pigmentation, associated with excision of the Tol2 element. The newly arising mutant gene is inherited in the Mendelian fashion. Thus, oculocutaneous albinism is not strictly irreversible, at least in this organism and the results also indicate that the insertion of the Tol2 element is the main, and possibly the only, cause of the i(b) albinism. Importantly our data also suggest that medaka fish possess an active transposase.