DNA-based Transposable Elements Research Articles

The medaka fish Tol2 transposable element is in an early stage of decay: identification of a nonautonomous copy.

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.

DNA-based transposable elements with nucleotide sequence similar to Tol2 from medaka fish are prevalent in cyprinid fishes

DNA-based transposable elements have been almost completely inactivated in vertebrate genomes. Tol2 is one of the few elements that were found in vertebrates and have been shown to be active. It is a member of the hAT (hobo/Activator/Tam3) transposable element family and was first identified in the medaka fish (Oryzias latipes) as an insertion sequence interrupting the tyrosinase gene of an albino mutant. Recently, an element named Tgf2 that exhibited a nucleotide identity as high as 97% with Tol2 was found in the goldfish (Carassius auratus), and its transposition activity was demonstrated. Goldfish is a species belonging to the order Cypriniformes, which is often represented by the common carp and includes zebrafish. Here, we surveyed 43 species of this order, with a focus on the Cyprinidae family, for the presence of Tol2-like sequences by PCR and Southern blot analyses. A wide distribution of such sequences was observed among cyprinid fishes. The hAT family elements generally tend to lose their internal regions over time, especially the central portion, by deletion mutations and all copies of the element in the genome are eventually shortened and inactivated. Our survey showed that approximately half of the cyprinid fishes examined retained this central region, suggesting that these elements have not been completely inactivated in the Cyprinidae family, and possibly also in the order Cypriniformes.

Horizontal transfer of a non-autonomous Helitron among insect and viral genomes.

BackgroundThe movement of mobile elements among species by horizontal transposon transfer (HTT) influences the evolution of genomes through the modification of structure and function. Helitrons are a relatively new lineage of DNA-based (class II) transposable elements (TEs) that propagate by rolling-circle replication, and are capable of acquiring host DNA. The rapid spread of Helitrons among animal lineages by HTT is facilitated by shuttling in viral particles or by unknown mechanisms mediated by close organism associations (e.g. between hosts and parasites).ResultsA non-autonomous Helitron independently annotated as BmHel-2 from Bombyx mori and the MITE01 element from Ostrinia nubilalis was predicted in the genomes of 24 species in the insect Order Lepidoptera. Integrated Helitrons retained ≥ 65% sequence identity over a 250 bp consensus, and were predicted to retain secondary structures inclusive of a 3′-hairpin and a 5′-subterminal inverted repeat. Highly similar Hel-2 copies were predicted in the genomes of insects and associated viruses, which along with a previous documented case of real-time virus-insect cell line transposition suggests that this Helitron has likely propagated by HTT.ConclusionsThese findings provide evidence that insect virus may mediate the HTT of Helitron-like TEs. This movement may facilitate the shuttling of DNA elements among insect genomes. Further sampling is required to determine the putative role of HTT in insect genome evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1318-6) contains supplementary material, which is available to authorized users.

Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

The Medaka zic1/zic4 Mutant Provides Molecular Insights into Teleost Caudal Fin Evolution

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.

Post-Genome Biology of Primates

Preface Ajit Varki 1. "Introduction" Yasuhiro Go, Hiroo Imai, and Hirohisa Hirai I. Post-Genomic Approaches toward Phenotype 2. Naoki Osada "An overview of transcriptome studies in nonhuman primates" 3. Mehmet Somel, Lin Tang, and Philipp Khaitovich "The role of neoteny in human evolution: from genes to the phenotype" 4. Yoshihito Niimura "Evolution of chemosensory receptor genes in primates and other mammals" 5. Kaylin A. Adipietro, Hiroaki Matsunami, and Hanyi Zhuang "Functional evolution of primate odorant receptors" 6. Tohru Sugawara and Hiroo Imai "Post genome biology of primates focusing on taste perception" 7. Shoji Kawamura, Chihiro Hiramatsu, Amanda D. Melin, Linda M. Fedigan, Filippo Aureli, and Colleen M. Schaffner "Polymorphic color vision in primates: evolutionary considerations" II. Genome Structure and its Applications 8. Toshiyuki Hayakawa and Ajit Varki "Human-specific changes in sialic acid biology" 9. Hiroki Oota and Kenneth K. Kidd "Duplicated gene evolution of the primate alcohol dehydrogenase family" 10. Yoko Satta "Genome structure and primate evolution" 11. Akihiko Koga "Contribution of DNA-based transposable elements to genome evolution: inferences drawn from behavior of an element found in fish" 12. Takashi Kitano "Application of phylogenetic network" III. Chromosome Genomics 13. Roscoe Stanyon, Nicoletta Archidiacono, and Mariano Rocchi "Comparative primate molecular cytogenetics: revealing ancestral genomes, marker order and evolutionary new centromeres" 14. Stefan Mueller and Johannes Wienberg "Chromosomal evolution of gibbons (Hylobatidae)" 15. Hirohisa Hirai "Evolution and biological meaning of genomic wastelands (RCRO): proposal of hypothesis" IV. Evolution of humans and Non-human Primates 16. Atsushi Matsui and Masami Hasegawa "Molecular phylogeny and evolution in primates" 17. Masanaru Takai "Origins and evolution of early primates"

Hen1 is required for oocyte development and piRNA stability in zebrafish

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2'-O-methyl modification at their 3' end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3'-5' exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.

Distribution of complete and defective copies of the Tol1 transposable element in natural populations of the medaka fish Oryzias latipes

DNA-based transposable elements are present in the genomes of various organisms, and generally occur in autonomous and nonautonomous forms, with a good correspondence to complete and defective copies, respectively. In vertebrates, however, the vast majority of DNA-based elements occur only in the nonautonomous form. Until now, the only clear exception known has been the Tol2 element of the medaka fish, which still causes mutations in genes of the host species. Here, we report another exception: the Tol1 element of the same species. This element was thought likely to be a "dead" element like the vast majority of vertebrate elements, but recent identification of an autonomous Tol1 copy in a laboratory medaka strain gave rise to the possibility that the element is still "alive" in medaka natural populations. We examined variation in the structure of Tol1 copies through genomic Southern blot analysis, and revealed that 10 of the 32 fish samples examined contained full-length Tol1 copies in their genomes. The frequency at which these copies occur among Tol1 copies is at most 0.5%, yet some of them still have the ability to produce a functional transposase. The medaka fish thus harbors two active DNA-based elements in its genome, and is in this respect unique among vertebrates.

Germline transgenesis of zebrafish using the medaka Tol1 transposon system

Tol1 is a DNA-based transposable element first identified from an albino mutant medaka fish. It has been demonstrated to function as an efficient gene transfer vector in mammalian cells. We now demonstrate Tol1 germline transgenesis in zebrafish. A construct containing the green fluorescence protein (GFP) reporter gene inserted between the Tol1 arms was microinjected together with Tol1 transposase mRNA into fertilized eggs. Sustained GFP expression was observed in 88% of 1-month-old fish, suggesting efficient transposon integration into somatic cells. Eleven of 24 adult GFP-positive fish yielded GFP-positive progeny. Sequencing analysis of Tol1 insertion sites in GFP-positive progeny confirmed Tol1 transposition-mediated integrations into zebrafish chromosomes. We also observed functional independence of the Tol1 transposase-substrate system from that of Tol2, another medaka-derived transposon. Coupled with its previously demonstrated maximal cargo capacity of >20 kb, Tol1 could serve as a useful addition to the zebrafish genetic engineering toolbox.

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.

The Tol1 element of medaka fish is transposed with only terminal regions and can deliver large DNA fragments into the chromosomes

Tol1 is an active DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes. This element belongs to the hAT transposable element family, of which complete copies have relatively long sequences. In addition, we found that Tol1 elements as long as 18 and 20 kb occur in the medaka fish genome. These facts suggest that Tol1 is suitable for carrying large DNA fragments as a gene transfer vector. Focusing on this, we conducted two kinds of manipulations of the element. The first was to eliminate internal regions dispensable for transposition. It was revealed that a Tol1 element consisting of 157-bp left- and 106-bp right-terminal regions could be transposed without a loss of transposition efficiency. Next, we prepared long Tol1 elements by incorporating unrelated DNA fragments into this short Tol1 clone and examined their transposition efficiencies. The transposition frequency decreased as the element size increased. The longest Tol1 element we examined measured 22.1 kb, and its transposition frequency was approximately one fifth that of a 2.1-kb element. However, this frequency was still significantly higher than that of a random integration of DNA into the chromosomes. The element size of 22.1 kb is the longest ever reported for DNA-based elements currently used for mammals. Thus, Tol1 is a superior gene-transfer vector with a large cargo capacity.

PCR detection of excision suggests mobility of the medaka fishTol1transposable element in the frogXenopus 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.

The Tol1 transposable element of the medaka fish moves in human and mouse cells

DNA-based transposable elements can be used as tools for gene engineering and gene therapy. A great advantage over RNA-mediated elements and retroviruses is the simplicity and safety of usage. The Tol1 element of the medaka fish Oryzias latipes has structural features of DNA-based elements. Although its excision has already been demonstrated, de novo insertion has not been observed, and a transposase has not been hitherto identified. We first cloned, through in silico search alignments and genomic library screenings, a 4.4-kb Tol1 copy carrying open reading frames and then identified, by mRNA analysis, a 2.9-kb transcript coding for 851 amino acids. The protein product of this transcript catalyzed transposition of a nonautonomous Tol1 copy in human and mouse culture cells. This identification of a fully functional Tol1 transposase could lead to the development of new tools for basic and translational molecular biology applications in mammals.

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.

The tyrosinase gene of the i(b) albino mutant of the medaka fish carries a transposable element insertion in the promoter region.

The i locus of the medaka fish contains the tyrosinase gene whose product is the key enzyme required for melanin biosynthesis. The i(b) allele at this locus, also denoted as i( 5), causes oculocutaneous albinism in homozygous carriers. Its albino phenotype is very weak, characterized mainly by small and varying sized melanophores in juveniles. Cloning and sequencing analyses of the tyrosinase gene for the i (b) allele revealed the presence of a 4.7-kb extra DNA fragment in the 5' untranslated region, this being Tol2, a DNA-based transposable element of the hobo Activator Tam3 (hAT) family which had previously been identified as a cause of another mutant allele i(4). Its insertion point was 85 bp upstream of the main transcription initiation site and 50 bp downstream of the CATGTG motif that has been suggested to be essential for the promoter function of the tyrosinase gene. The transcription level of the tyrosinase gene was decreased in i(b)/i(b) fish, compared with wild-type fish. The insertion is thus a likely cause of the weak albino phenotype. The Tol2 element transposes in a cut-and-paste fashion, and its excision is mostly imprecise, leaving some nucleotides and/or removing excess nucleotides. The i (b) mutant strain can thus be expected to serve as a source from which various other mutations in the promoter region can be derived.

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.

Transposable elements in medaka fish.

DNA-based transposable elements appear to have been nearly or completely inactivated in vertebrates. Therefore the elements of the medaka fish Oryzias latipes that still have transposition activity provide precious materials for studying transposition mechanisms, as well as the evolution, of transposable elements in vertebrates. Fortunately, the medaka fish has a strong background for genetic and evolutionary studies. The advantages of this host species and their elements, together with results so far obtained, are here described.

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.

Targeting transposition: at home in the genome.

The completion of the Saccharomyces genome sequence in 1996 signaled the beginning of a new chapter in repetitive element sequence analysis (Cherry et al. 1997). The genomic analysis in the article by Kim et al. (1998) in this issue provides new insights into the nature and distribution of repetitive DNA sequences in the Saccharomyces genome. It not only assesses the repetitive sequences present in the genome but also clarifies what is not found. Saccharomyces has five long terminal repeat (LTR)– retrotransposon families (Ty elements) but no nuclear LINE-like or SINE retroelements and no identifiable DNA-based transposable elements. Remarkably, each Ty family displays insertion specificity. The parallels and differences between the organization of transposable elements in the Saccharomyces genome, and what is thus far known of the organization of elements in the genomes of other eukaryotes, suggest that this chapter will have many interesting sequelae. The genomic analysis by Kim et al. (1998) further defines the relationships of the Ty families of yeast comprised of four copia-like (Ty1, Ty2, Ty4, and Ty5) and one gypsy-like (Ty3) families. The copia-like families have been divided further based on similarity of encoded proteins and LTRs. Ty1 and Ty2 share significant similarity, particularly in the capsid domain, and share a common LTR, called d. In addition, Kim et al. showed for the first time, that Ty1 and Ty2 d elements are distinct—differing consistently at a single position. Unlike retrovirus LTRs, yeast element LTRs flanking the internal domain undergo recombination, thereby deleting one copy of the LTR and the internal domain sequence. Interestingly, among the families, there are widely differing copy numbers of complete elements and LTRs: 32 Ty1 elements and 217 Ty1-type d elements; 13 Ty2 elements and 34 Ty2type d elements; two Ty3 elements and 41 s elements; 3 Ty4 elements and 32 t elements; and one Ty5 element and seven v elements. Thus, ratios of LTR sequences to complete elements range from 7 for Ty1 to 21 for Ty3. Because there are many more Ty1 insertions than Ty3 insertions and because the Ty1-type d elements are more degenerate than the s elements, Ty1 probably represents an older class of elements within yeast. The fact that the ratio of LTR to complete copies is so much higher for Ty3 suggests that generation of isolated LTRs occurs more frequently for some sequences than for others. With stunning completeness, the genome sequence reveals the nonrandom nature of the retroelement insertions for each family (see Fig. 1) (Chalker and Sandmeyer 1992; Devine and Boeke 1996; Zou et al. 1996; Bryk et al. 1997; Smith and Boeke 1997). In addition to tRNA genes, 5S, U6, and PI RNA genes were also found associated with insertions. The Tyl, Ty2, Ty3, and Ty4 elements target the upstream region of RNA polymerase 111-transcribed genes: 196 of 217 Ty1; 28 of 34 Ty2; 40 of 41 Ty3; and 30 of 32 Ty4 insertions are within 750 bp of a gene transcribed by RNA polymerase III. This listing is conservative because it does not exclude the possibility that some of the seemingly exceptional insertions are associated with unidentified targets, such as unknown or degenerate polymerase IIItranscribed genes. Ty5 does not target tRNA genes. Instead, Ty5 inserts into silenced regions of the genome, the telomeres and mating type loci. In addition to these important insights, the analysis of Kim et al. (1998) raises provocative questions concerning the relationship between repetitive elements and their habitats: Why is Saccharomyces apparently lacking major classes of elements, including quite ancient ones found in bacteria; what is the impact of these elements on genome maintenance; and why do these elements target particular genomic regions? These questions are addressed in the remainder of this article.