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.     

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.     

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.     

Detection of de novo insertion of the medaka fish transposable element Tol2

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.     

Evidence for recent invasion of the medaka fish genome by the Tol2 transposable element

Tol2 is a transposable element of the terminal-inverted-repeat class, residing in the genome of the medaka fish Oryzias latipes. The genus Oryzias contains more than 10 species for which phylogenetic relationships have previously been estimated. To infer the history of Tol2 in this genus we performed genomic Southern blots and PCR analyses of 10 of the species. It was revealed that Tol2 occurs in 2 of the 10 species (O. curvinotus and O. latipes) and that the length and the restriction map structure of Tol2 are identical in the two cases. Further, sequencing analysis revealed an extremely low level of divergence compared with that in a nuclear gene. These results suggest recent incorporation of Tol2 into one or both of the two species, implying horizontal transfer of Tol2 from one species to the other or into them both from a common source.     

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.     

Heparan sulfate proteoglycans exert positive and negative effects in Shh activity

Hedgehog (Hh) proteins are morphogens involved in short- and long-range effects during early embryonic development. Genetic analysis in fly and vertebrate embryos showed that heparan sulfate proteoglycans (HSPGs) are required for Hh transport and signaling. To further understand how HSPGs regulate Sonic hedgehog (Shh), we performed experiments using cell culture and biochemical assays. When the synthesis of HSPGs was reduced, a decrease in Shh activity was observed. Contrary to that, addition of a peptide that competes the binding of Shh to HSPGs resulted in augmentation of Shh activity. From these results, we concluded that HSPGs exert positive and negative effects in Shh activity. This dual effect correlates with the finding that Shh interacts preferentially with two HSPGs. The current model for the role of HSPGs in Shh diffusion is discussed in view of our findings.     

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.     

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.     

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.     

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.     

Phenotypic manifestations of yeast transposon insertion in the LYS2 gene and deletions resulting from imprecise excision of the transposon

The lys2-32 mutant allele resulted from Ty1 element insertion was identified and cloned. The expression and reversions of lys2-32 localized in an autonomous plasmid were studied. The insertion was shown to inactivate LYS2 gene incompletely. Spontaneous reversions to complete or almost complete prototrophy were also obtained. About 50% of revertants retained the insertion. Others arise as a result of imprecise excision events leading to deletions of adjacent LYS2 sequences.     

Antimutagenic effects of chemicals on mutagenesis resulting from excision of a transposon in Salmonella typhimurium

We have found that a temperature-sensitive mutation in the polA gene of Salmonella typhimurium strain LT2 causes precise excision of transposon Tn10 to occur at significantly increased frequencies in cells incubated at the restrictive temperature. In our experiments, precise excision from a site in the tryptophan operon was measured by determining the frequency of reversion of the auxotrophic trp1014::Tn10 polA7 strain to prototrophy on defined medium containing a trace amount of broth. Because the yields of revertants at 37 degrees C were of the order of 200 colonies per plate, it was possible to measure the effects of chemical inhibitors on the processes involved in precise excision. We now report that all of the DNA-repair inhibitors we have studied (caffeine, ethionine, acriflavine, procaine and cinnamaldehyde) are effective inhibitors of precise excision of Tn10, and can therefore be defined as antimutagens.     

iTRAQ-based proteomic study of the effects of microcystin-LR on medaka fish liver

Microcystins are cyanotoxins that occur in ground water and thus pose a potential health risk. Microcystin-LR (microcystin-leucine-arginine) is a potent hepatotoxin, and is suspected of being a tumour promoter. Poisoning with this toxin causes several dysfunctions in hepatocytes by inhibiting protein phosphatases 1 and 2A, and notably produces oxidative stress, disrupts the cytoskeleton, and deregulates mitogen-activated protein kinase pathway. Medaka fish (Oryzias latipes) was chosen as a model for studying the effects of this cyanotoxin on liver proteins using a gel-free approach, iTRAQ. Fish were gavaged with microcystin-LR. Two hours later, 325 proteins could be identified by Scaffold Q+ and 32 proteins revealed statistically significant variations above a ∣0.2∣ threshold of log(2) ratio by comparison with control. These proteins are mostly involved in the translation and maturation of proteins, lipid metabolism and detoxification. Notably, apolipoproteins are deregulated which indicates a possible alteration of chylomicron-mediated transport.     

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.     

Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol-1 transposable element from the tyrosinase gene

The medaka fish albino mutant, i(1) is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol-1. Recently, mosaic black-pigmented i(1) medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol-1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.     

TGF-beta signaling: positive and negative effects on tumorigenesis.

TGF-beta binding to the cell surface triggers activation of multiple signal transduction pathways that are connected in intricate ways with each other, and with other response networks involved in sensing cellular information input. Recent data indicate that changes in signal intensity and connectivity of these pathways may underlie the complex transition of the TGF-beta pathway from tumor suppressor to oncogene during tumorigenesis.     

Uncoupling of omnivore-mediated positive and negative effects on periphyton mats

The riverine grass shrimp (Palaemonetes paludosus) and eastern mosquitofish (Gambusia holbrooki) consume periphyton and small invertebrates, potentially affecting periphyton through negative effects (i.e., consumption) and/or positive effects such as nutrient regeneration, physical stimulation, and trophic cascades. We performed field experiments in the Everglades in which omnivores and periphyton were maintained in cages, with a fraction of the periphyton held in omnivore-exclusion bags that allowed passage of nutrients but prevented its consumption or physical disturbance. In some instances, periphyton growth rate increased with increasing omnivore biomass. Omnivores probably stimulated periphyton growth through nutrient regeneration, possibly subsidizing periphyton with nutrients derived from ingested animal prey. The net balance of omnivore-mediated negative and positive effects varied among experiments because of seasonal and spatial differences in periphyton characteristics. Consumption of periphyton mats might have been reduced by the arrangement of palatable algae (green algae and diatoms) within a matrix of unpalatable ones (CaCO(3)-encrusting filamentous cyanobacteria). In a laboratory feeding experiment, mosquitofish consumed more green algae and diatoms in treatments with disrupted mat structure than in those with intact mats. No difference in diet was observed for shrimp. Our study underscores the complexity of consumer-periphyton interactions in which periphyton edibility affects herbivory and consumers influence periphyton through multiple routes that cannot be fully appreciated in experiments that only investigate net effects.