Molecular characterization of Vulmar1, a complete mariner transposon of sugar beet and diversity of mariner- and En/Spm-like sequences in the genus Beta.

Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.     

Structure and evolution of the hAT transposon superfamily.

The maize transposon Activator (Ac) was the first mobile DNA element to be discovered. Since then, other elements were found that share similarity to Ac, suggesting that it belongs to a transposon superfamily named hAT after hobo from Drosophila, Ac from maize, and Tam3 from snapdragon. We addressed the structure and evolution of hAT elements by developing new tools for transposon mining and searching the public sequence databases for the hallmarks of hAT elements, namely the transposase and short terminal inverted repeats (TIRs) flanked by 8-bp host duplications. We found 147 hAT-related sequences in plants, animals, and fungi. Six conserved blocks could be identified in the transposase of most hAT elements. A total of 41 hAT sequences were flanked by TIRs and 8-bp host duplications and, out of these, 34 sequences had TIRs similar to the consensus determined in this work, suggesting that they are active or recently active transposons. Phylogenetic analysis and clustering of hAT sequences suggest that the hAT superfamily is very ancient, probably predating the plant-fungi-animal separation, and that, unlike previously proposed, there is no evidence that horizontal gene transfer was involved in the evolution of hAT elements.     

Phylogenetic and functional characterization of the hAT transposon superfamily

Transposons are found in virtually all organisms and play fundamental roles in genome evolution. They can also acquire new functions in the host organism and some have been developed as incisive genetic tools for transformation and mutagenesis. The hAT transposon superfamily contains members from the plant and animal kingdoms, some of which are active when introduced into new host organisms. We have identified two new active hAT transposons, AeBuster1, from the mosquito Aedes aegypti and TcBuster from the red flour beetle Tribolium castaneum. Activity of both transposons is illustrated by excision and transposition assays performed in Drosophila melanogaster and Ae. aegypti and by in vitro strand transfer assays. These two active insect transposons are more closely related to the Buster sequences identified in humans than they are to the previously identified active hAT transposons, Ac, Tam3, Tol2, hobo, and Hermes. We therefore reexamined the structural and functional relationships of hAT and hAT-like transposase sequences extracted from genome databases and found that the hAT superfamily is divided into at least two families. This division is supported by a difference in target-site selections generated by active transposons of each family. We name these families the Ac and Buster families after the first identified transposon or transposon-like sequence in each. We find that the recently discovered SPIN transposons of mammals are located within the family of Buster elements.     

An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.     

Mx-rMx, a family of interacting transposons in the growing hAT superfamily of maize

More than half a century after the discovery of transposable elements, the number of genetically defined autonomous elements that have been isolated and characterized molecularly in any one species remains surprisingly small. Because of its rich genetic history, maize (Zea mays) is, by far, the plant with the largest number of such elements. Yet, even in maize, a maximum of only two autonomous elements have been characterized in any transposon superfamily. This article describes the isolation and molecular and genetic characterization of Mx (for mobile element induced by x-rays), a third autonomous member of the hAT transposon superfamily in maize. Mx is 3731 bp long, ends in 13-bp terminal inverted repeats (TIRs), and causes an 8-bp duplication of the target site. Mx and rMx (for responder to Mx), its 571-bp nonautonomous partner, define a classical family of interacting transposable elements. Surprisingly, the TIRs of Mx and rMx are only 73% identical, and the subterminal sequences are even less so, suggesting that Mx and rMx may represent diverging transposable elements still capable of mobilization by the same transposase. Sequences that are closer to the ends of either Mx or rMx are present in the maize genome. Mx is predicted to encode a 674-amino acid protein that is homologous to the Ac transposase. Although Mx and Ac are closely related, they do not interact. Other data suggest that maize may possess at least five families of hAT transposons that do not interact with each other. The possible origin of noninteracting transposon families within the same superfamily is discussed.     

A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ～ 160 Mb) has been sequenced and is composed of ～65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.     

Recombination technologies for enhanced transgene stability in bioengineered insects

Transposon-based vectors currently provide the most suitable gene transfer systems for insect germ-line transformation and are used for molecular improvement of the Sterile Insect Technique. However, the long time stability of genome-integrated transposon constructs depends on the absence of transposase activity that could remobilize the transposon-embedded transgenes. To achieve transgene stability transposon vectors are usually non-autonomous, lacking a functional transposase gene, and chosen so that endogenous or related transposon activities are not present in the host. Nevertheless, the non-autonomous transposon-embedded transgenes could become unstable by the unintended presence of a mobilizing transposase that may have been undetected or subsequently entered the host species by horizontal gene transfer. Since the field release of transgenic insects will present environmental concerns relating to large populations and high mobility, it will be important to ensure that transgene constructs are stably integrated for maintaining strain integrity and eliminating the possibility for unintentional transfer into the genome of another organism. Here we review efficient methods to delete or rearrange terminal repeat sequences of transposons necessary for their mobility, subsequent to their initial genomic integration. These procedures should prevent transposase-mediated remobilization of the transgenes, ensuring their genomic stability.     

The hAT family: a versatile transposon group common to plants, fungi, animals, and man

Transposons are ubiquitous mobile genetic elements found in all eu- and prokaryotic cells. The first transposon identified, the maize Activator element, belongs to the hAT family. hAT transposons have been identified in most eukaryotic lineages, including plants, fungi, animals and even man. The basic structural and functional features of this transposon family and its phylogenetic roots are discussed in detail, including a phylogenetic tree deduced from the amino acid sequence of the most conserved part of the transposon-encoded transposase. Emphasis is given to the use of hAT transposons as tools for gene tagging and insect transformation as well as to their biological function, i.e. are they selfish DNA, beneficial companions, or even both?     

The <Emphasis Type="Italic">Tc1/mariner</Emphasis> DNA transposons in the genome of mollusk <Emphasis Type="Italic">Littorina saxatilis</Emphasis>

The Tc1/mariner superfamily is one of the most widely distributed among the DNA transposons in both terrestrial and aquatic organisms. We studied the abundance of the Tc1/mariner elements in the genome of the gastropod Littorina saxatilis Olivi, 1792 (Gastropoda: Littorinimorpha). For this purpose, nucleotide sequences with a total length of 358877 bp were analyzed. Six sequences were found to be similar to the Tc1/mariner DNA transposons. These sequences were studied for structure, the presence of functional transposase, and the systematic position within the superfamily. In addition, the loci with high homology to the DNA transposons of the hAT, Sola, Ginger, EnSpm/CACTA, ISL2EU, Kolobok, Novosib, Zisupton, and Helitron superfamilies were identified.     

Zisupton--a novel superfamily of DNA transposable elements recently active in fish

Transposable elements are widespread mobile DNA sequences able to integrate into new locations within genomes. Through transposition and recombination, they significantly contribute to genome plasticity and evolution. They can also regulate gene expression and provide regulatory and coding sequences (CDSs) for the evolution of novel gene functions. We have identified a new superfamily of DNA transposon on the Y chromosome of the platyfish Xiphophorus maculatus. This element is 11 kb in length and carries a single CDS of 24 exons. The N-terminal part of the putative protein, which is expressed in all adult tissues tested, contains several nucleic acid- and protein-binding domains and might correspond to a novel type of transposase/integrase not described so far in any transposon. In addition, a testis-specific splice isoform encodes a C-terminal Ulp1 SUMO protease domain, suggesting a function in posttranslational protein modification mediated by SUMO and/or ubiquitin small peptides. Accordingly, this element was called Zisupton, for Zinc finger SUMO protease transposon. Beside the Y-chromosomal sequence, five other very similar copies were identified in the platyfish genome. All copies are delimited by 99-bp conserved subterminal inverted repeats and flanked by copy-specific 8-nt target site duplications reflecting their integration at different positions in the genome. Zisupton elements are inserted at different genomic locations in different poeciliid species but also in different populations of X. maculatus. Such insertion polymorphisms between related species and populations indicate relatively recent transposition activity, with a high degree of nucleotide identity between species suggesting possible implication of horizontal gene transfer. Zisupton sequences were detected in other fish species, in urochordates, cephalochordates, and hemichordates as well as in more distant organisms, such as basidiomycete fungi, filamentous brown algae, and green algae. Possible examples of nuclear genes derived from Zisupton have been identified. To conclude, our analysis has uncovered a new superfamily of DNA transposons with potential roles in genome diversity and evolutionary innovation in fish and other organisms.     

Identification of active transposon dTok, a member of the hAT family, in rice

Recent completion of the sequencing of the rice genome has revealed that it contains >40% repetitive sequences, most of which are related to inactive transposable elements. During the molecular analysis of the floral organ number 1/multiple pistil 2 (fon1/mp2) mutant, we identified an active transposable element dTok0 that was inserted at the kinase domain of FON1, a homolog of CLAVATA1. Insertion of the element into FON1 generated an 8 bp duplication of its target sites, which is one of the major characteristics of the hAT family of transposons. The dTok0 element was actively transposed out of the FON1 gene, leaving 5-8 bp footprints. Reinsertion into a new location was observed at a low frequency. Analysis of the genome sequence showed that the rice cultivar 'Nipponbare' contains 25 copies of dTok elements; similar numbers were present in all the Oryza species examined. Because dTok0 does not encode a transposase, enzyme activity should be provided in trans. We identified a putative autonomous transposon, Tok1 that contains an intact open reading frame of the Ac-like transposase.     

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.     

Horizontal Escape of the Novel Tc1-Like Lepidopteran Transposon TCp3.2 into Cydia pomonella Granulovirus

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons. Received: 27 February 1997 / Accepted: 16 May 1997     

MMTS, a new subfamily of Tc1-like transposons

A novel Tc1-like transposable element has been identified as a new DNA transposon in the mud loach, Misgurnus mizolepis. The M. mizolepis Tc1-like transposon (MMTS) is comprised of inverted terminal repeats and a single gene that codes Tc1-like transposase. The deduced amino acid sequence of the transposase-encoding region of MMTS transposon contains motifs including DDE motif, which was previously recognized in other Tc1-like transposons. However, putative MMTS transposase has only 34-37% identity with well-known Tc1, PPTN, and S elements at the amino acid level. In dot-hybridization analysis used to measure the copy numbers of the MMTS transposon in genomes of the mud loach, it was shown that the MMTS transposon is present at about 3.36 x 104 copies per 2 x 109 bp, and accounts for approximately 0.027% of the mud loach genome. Here, we also describe novel MMTS-like transposons from the genomes of carp-like fishes, flatfish species, and cichlid fishes, which bear conserved inverted repeats flanking an apparently intact transposase gene. Additionally, BLAST searches and phylogenetic analysis indicated that MMTS-like transposons evolved uniquely in fishes, and comprise a new subfamily of Tc1-like transposons, with only modest similarity to Drosophila melanogaster (foldback element FB4, HB2, HB1), Xenopus laevis, Xenopus tropicalis, and Anopheles gambiae (Frisky).     

Identification of Novel Non-autonomous CemaT Transposable Elements and Evidence of their Mobility within the C. elegans Genome

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152–367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4’s ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.     

CemaT1 is an active transposon within the Caenorhabditis elegans genome

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. In the nematode Caenorhabditis elegans, there are eight copies of CemaT1 that are predicted to encode a functional transposase, with five copies being >99% identical. We present evidence, based on searches of publicly available databases and on PCR-based mobility assays, that the CemaT1 transposase is expressed in C. elegans and that the CemaT transposons are capable of excising in both somatic and germline tissues. We also show that the frequency of CemaT1 excisions within the genome of the N2 strain of C. elegans is comparable to that of the Tc1 transposon. However, unlike Tc transposons in mutator strains of C. elegans, maT transposons do not exhibit increased frequencies of mobility, suggesting that maT is not regulated by the same factors that control Tc activity in these strains. Finally, we show that CemaT1 transposons are capable of precise transpositions as well as orientation inversions at some loci, and thereby become members of an increasing number of identified active transposons within the C. elegans genome.     

The ancient mariner sails again: transposition of the human Hsmar1 element by a reconstructed transposase and activities of the SETMAR protein on transposon ends

Hsmar1, one of the two subfamilies of mariner transposons in humans, is an ancient element that entered the primate genome lineage approximately 50 million years ago. Although Hsmar1 elements are inactive due to mutational damage, one particular copy of the transposase gene has apparently been under selection. This transposase coding region is part of the SETMAR gene, in which a histone methylatransferase SET domain is fused to an Hsmar1 transposase domain. A phylogenetic approach was taken to reconstruct the ancestral Hsmar1 transposase gene, which we named Hsmar1-Ra. The Hsmar1-Ra transposase efficiently mobilizes Hsmar1 transposons by a cut-and-paste mechanism in human cells and zebra fish embryos. Hsmar1-Ra can also mobilize short inverted-repeat transposable elements (MITEs) related to Hsmar1 (MiHsmar1), thereby establishing a functional relationship between an Hsmar1 transposase source and these MITEs. MiHsmar1 excision is 2 orders of magnitude more efficient than that of long elements, thus providing an explanation for their high copy numbers. We show that the SETMAR protein binds and introduces single-strand nicks into Hsmar1 inverted-repeat sequences in vitro. Pathway choices for DNA break repair were found to be characteristically different in response to transposon cleavage mediated by Hsmar1-Ra and SETMAR in vivo. Whereas nonhomologous end joining plays a dominant role in repairing excision sites generated by the Hsmar1-Ra transposase, DNA repair following cleavage by SETMAR predominantly follows a homology-dependent pathway. The novel transposon system can be a useful tool for genome manipulations in vertebrates and for investigations into the transpositional dynamics and the contributions of these elements to primate genome evolution.     

A hAT-related family of interspersed repetitive elements in genomes of western Palaearctic water frogs

A family of interspersed repetitive elements, RlBamHI, with sequence similarity to the transposase of hAT DNA transposons, occurs in genomes of eight western Palaearctic water frog taxa and the brown frog Rana dalmatina , but was not detected in Xenopus laevis or Salamandra salamandra . RlBamHI elements are not tandemly arrayed, are dispersed across all chromosomes although not uniformly distributed, and based on dot-blot hybridizations may constitute as much as approximately 10% of the genomes of Rana lessonae , Rana ridibunda , and Rana perezi , but only approximately 1% of that of Rana saharica . Eleven nucleotide sequences of a 572 bp fragment from the nine taxa are very similar (pairwise differences 0.4–8.1% for nucleotides, 0.6–18.2% for amino acids), and all share a single open reading frame across the whole RlBamHI fragment. The reading frame is maintained despite several indels, most of which are multiples of 3 bp, but a pair of which in one species alters and, after 13 codons, restores the reading frame. It is possible that the reading frame is selectively maintained, suggesting recent or even present transposition capacity. The amino acid sequences encoded by RlBamHI elements, but not the nucleotide sequences themselves, reveal the similarity of RlBamHI to members of the widespread hAT superfamily of DNA transposons. Amino acid sequence comparisons permitted no convincing phylogenetic placement of RlBamHI among 32 representative hAT transposons across organisms, probably reflecting both the genetic divergence of RlBamHI elements and the paucity of aligned residues available for analysis. Among DNA transposons, a genomic fraction of 10% is extraordinarily high. Phylogenetic analyses of Rana RlBamHI sequences match poorly with independent genetic and molecular phylogenies; the elements compared probably are not orthologous, which renders their sequences inadequate for reconstructing organismal phylogenetic histories.