Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera

Transposable elements of the mariner family are widespread among insects and other invertebrates, and initial analyses of their relationships indicated frequent occurrence of horizontal transfers between hosts. A specific PCR assay was used to screen for additional members of the irritans subfamily of mariners in more than 400 arthropod species. Phylogenetic analysis of cloned PCR fragments indicated that relatively recent horizontal transfers had occurred into the lineages of a fruit fly Drosophila ananassae, the horn fly Haematobia irritans, the African malaria vector mosquito Anopheles gambiae, and a green lacewing Chrysoperla plorabunda. Genomic dot-blot analysis revealed that the copy number in these species varies widely, from about 17,000 copies in the horn fly to three copies in D. ananassae. Multiple copies were sequenced from genomic clones from each of these species and four others with related elements. These sequences confirmed the PCR results, revealing extremely similar elements in each of these four species (greater than 88% DNA and 95% amino acid identity). In particular, the consensus sequence of the transposase gene of the horn fly elements differs by just two base pairs out of 1,044 from that of the lacewing elements. The mosquito lineage has diverged from the other Diptera for over 200 Myr, and the neuropteran last shared a common ancestor with them more than 265 Myr ago, so this high similarity implies that these transposons recently transferred horizontally into each lineage. Their presence in only the closest relatives in at least the lacewing lineage supports this hypothesis. Such horizontal transfers provide an explanation for the evolutionary persistence and widespread distribution of mariner transposons. We propose that the ability to transfer horizontally to new hosts before extinction by mutation in the current host constitutes the primary selective constraint maintaining the sequence conservation of mariners and perhaps other DNA-mediated elements.      

A potentially functional mariner transposable element in the protist Trichomonas vaginalis

Mariner transposable elements encoding a D,D34D motif-bearing transposase are characterized by their pervasiveness among, and exclusivity to, animal phyla. To date, several hundred sequences have been obtained from taxa ranging from cnidarians to humans, only two of which are known to be functional. Related transposons have been identified in plants and fungi, but their absence among protists is noticeable. Here, we identify and characterize Tvmar1, the first representative of the mariner family to be found in a species of protist, the human parasite Trichomonas vaginalis. This is the first D,D34D element to be found outside the animal kingdom, and its inclusion in the mariner family is supported by both structural and phylogenetic analyses. Remarkably, Tvmar1 has all the hallmarks of a functional element and has recently expanded to several hundred copies in the genome of T. vaginalis. Our results show that a new potentially active mariner has been found that belongs to a distinct mariner lineage and has successfully invaded a nonanimal, single-celled organism. The considerable genetic distance between Tvmar1 and other mariners may have valuable implications for the design of new, high-efficiency vectors to be used in transfection studies in protists.     

转座元件mariner

自mariner转座元件在Drosophila mauritiana中首次发现至今已经在包括人类在内的多种生物体中证实了mariner及类mariner元件（MLEs）的存在。MLEs属于mariner/Tc1超家族-II型转座元件中分布最广、种类最多的超家族之一。MLEs的转座酶都具有“D,D(34)D”的结构,并能催化MLEs通过“剪切和粘贴”机制进行转座。它们的宿主广泛和多样,能够进行种系传递,这都表明MLEs的转座不需要宿主特异元件的参与。 MLEs对多种生物尤其对脊椎动物的成功转化更支持了它们的不依赖宿主的转座机制,而且让人们看到了它们作为转基因载体的巨大潜能。 Abstract: Mariner and mariner-like elements (MLEs) have been found in a wide range of organisms including human since its discovery in Drosophila mauritiana. MLEs belong to the mariner/Tc1 superfamily, one of the most diverse and widespread Class II transposable elements. MLEs have a conserved “D,D(34)D” motif in their transposases and they transpose by cut-and-paste mechanisms. Their extraordinarily wide host range and horizontal transmission in distantly related species indicate that they do not need additional host-specific factors for transposition. The evidence that MLEs could transform a wide variety of organisms especially the vertebrates supported the host-independent mechanism and suggested the availability as a kind of potential transforming vector.     

A purified mariner transposase is sufficient to mediate transposition in vitro.

Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5' and 3' cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.     

Regulation of the transposable element mariner

The mariner/ Tc1 superfamily of transposable elements is widely distributed in animal genomes and is especially prevalent in insects. Their wide distribution results from their ability to be disseminated among hosts by horizontal transmission and also by their ability to persist in genomes through multiple speciation events. Although a great deal is known about the molecular mechanisms of transposition and excision, very little is known about the mechanisms by which transposition is controlled within genomes. The issue of mariner/Tc1 regulation is critical in view of the great interest in these elements as vectors for germline transformation of insect pests and vectors of human disease. Several potentially important regulatory mechanisms have been identified in studies of genetically engineered mariner elements. One mechanism is overproduction inhibition, in which excessive wild-type transposase reduces the rate of excision of a target element. A second mechanism is mediated by certain mutant transposase proteins, which antagonize the activity of the wild-type transposase. The latter process may help explain why the vast majority of MLEs in nature undergo ‘vertical inactivation’ by multiple mutations and, eventually, stochastic loss. Another potential mechanism of regulation may result from transposase titration by defective elements that retain their DNA binding sites and ability to transpose. There is also evidence that some mariner/Tc1 elements can be mobilized in a type of hybrid dysgenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Promiscuous target interactions in the mariner transposon Himar1

We have previously characterized the early intermediates of mariner transposition. Here we characterize the target interactions that occur later in the reaction. We find that, in contrast to the early transposition intermediates, the strand transfer complex is extremely stable and difficult to disassemble. Transposase is tightly bound to the transposon ends constraining rotation of the DNA at the single strand gaps in the target site flanking the element on either side. We also find that although the cleavage step requires Mg2+ or Mn2+ as cofactor, the strand transfer step is also supported by Ca2+, suggesting that the structure of the active site changes between cleavage and insertion. Finally, we show that, in contrast to the bacterial cut and paste transposons, mariner target interactions are promiscuous and can take place either before or after cleavage of the flanking DNA. This is similar to the behavior of the V(D)J system, which is believed to be derived from an ancestral eukaryotic transposon. We discuss the implications of promiscuous target interactions for promoting local transposition and whether this is an adaptation to facilitate the invasion of a genome following horizontal transfer to a new host species.     

Recent horizontal transfer of mellifera subfamily mariner transposons into insect lineages representing four different orders shows that selection acts only during horizontal transfer

We report the isolation and sequencing of genomic copies of mariner transposons involved in recent horizontal transfers into the genomes of the European earwig, Forficula auricularia; the European honey bee, Apis mellifera; the Mediterranean fruit fly, Ceratitis capitata; and a blister beetle, Epicauta funebris, insects from four different orders. These elements are in the mellifera subfamily and are the second documented example of full-length mariner elements involved in this kind of phenomenon. We applied maximum likelihood methods to the coding sequences and determined that the copies in each genome were evolving neutrally, whereas reconstructed ancestral coding sequences appeared to be under selection, which strengthens our previous hypothesis that the primary selective constraint on mariner sequence evolution is the act of horizontal transfer between genomes.     

Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.     

Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Genetic and molecular evidence presented in this paper demonstrate that the Mos factor for inherited mosaicism is a special copy of the transposable element mariner. Mosaicism observed in the presence of the Mos (Mosaic) factor results from a high frequency of excision of the mariner element from an insertion site near the white-eye gene in Drosophila mauritiana. The Mos factor promotes the excision of mariner elements from genomic insertion sites other than the site in wpch, and it also promotes its own loss from the genome. Putative transpositions of Mos to new genomic sites have also been observed. A copy of mariner present at a particular site in a Mos strain has been shown to be missing in derived strains in which the Mos factor has been lost, and in strains with putative transpositions. We propose that this copy of mariner is identical to the Mos factor.     

Selective constraints on P-element evolution.

P elements, like mariners, inhabit eukaryotic genomes and transpose via a DNA intermediate. Mutant and wild-type elements in the same genome should be transposed with equal probability by trans-acting transposase, and so no selection should counteract the accumulation of inactivating mutations in transposase genes. Thus, copies of mariner elements diverge within a host species under no selection (Robertson and Lampe 1995). It is unknown whether or not this pattern holds for P elements, which are unrelated to mariner elements but share the same life history. Publicly available P-element sequences were analyzed for evidence of conservative selection for the function of P-element-encoded proteins. Results were compared to predictions derived from several hypotheses that could explain selection, or the lack of it. P-element protein-coding sequences do evolve under conservative selection but apparently because of more than one selective force. Of the four exons in the P-element transposase, the first three (exons 0, 1, and 2) can be translated alone into a repressor of transposition, while the last (exon 3) is only expressed as part of the full-length transposase and probably serves a transposition-specific role. As full-length P-element copies diverge from each other within a host population, selection maintains exons 0-2 but apparently not exon 3. The selection acting on exons 0-2 may act at the host level for repression of transposition (since host level selection does act on orthologous truncated elements that contain only exons 0-2). Evidence of selection on exon 3 is only found in comparisons of more diverged elements from different species, suggesting that selection for transposition acts primarily at horizontal transfer events. Thus, horizontal transfer events may be the sole source of the selection that is crucial to the maintenance of autonomous P elements in the face of mutation (as suggested by Robertson and Lampe 1995). The predictions derived here suggest a strategy for collecting sequence data that could definitively answer these questions.     

Self-inflicted wounds, template-directed gap repair and a recombination hotspot. Effects of the mariner transposase

Aberrant repair products of mariner transposition occur at a frequency of approximately 1/500 per target element per generation. Among 100 such mutations in the nonautonomous element peach, most had aberrations in the 5' end of peach (40 alleles), in the 3' end of peach (11 alleles), or a deletion of peach with or without deletion of flanking genomic DNA (29 alleles). Most mariner mutations can be explained by exonuclease "nibble" and host-mediated repair of the double-stranded gap created by the transposase, in contrast to analogous mutations in the P element. In mariner, mutations in the 5' inverted repeat are smaller and more frequent than those in the 3' inverted repeat, but secondary mutations in target elements with a 5' lesion usually had 3' lesions resembling those normally found at the 5' end. We suggest that the mariner transposase distinguishes between the 5' and 3' ends of the element, and that the 5' end is relatively more protected after strand scission. We also find: (1) that homolog-dependent gap repair is a frequent accompaniment to mariner excision, estimated as 30% of all excision events; and (2) that mariner is a hotspot of recombination in Drosophila females, but only in the presence of functional transposase.     

Excision of the Drosophila mariner transposon Mos1. Comparison with bacterial transposition and V(D)J recombination

It has been proposed that the modern immune system has evolved from a transposon in an ancient vertebrate. While much is known about the mechanism by which bacterial transposable elements catalyze double-strand breaks at their ends, less is known about how eukaryotic transposable elements carry out these reactions. We have examined the mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage. We show that the nontransferred strand is cleaved initially, unlike prokaryotic transposons which cleave the transferred strand first. First strand cleavage is not tightly coupled to second strand cleavage and can occur independently of synapsis, as happens in V(D)J recombination but not in transposition of prokaryotic transposons. Unlike V(D)J recombination, however, second strand cleavage of mariner does not occur via a hairpin intermediate.     

A New Basal Subfamily of mariner Elements in Ceratitis rosa and Other Tephritid Flies

Several copies of highly related transposable elements, Crmar2, Almar1, and Asmar1, are described from the genomes of Ceratitis rosa, Anastrepha ludens, and A. suspensa, respectively. One copy from C. rosa, Crmar2.5, contains a full-length, uninterrupted ORF. All the other copies, from the three species contain a long deletion within the putative ORF. The consensus Crmar2 element has features typical of the mariner/Tc1 superfamily of transposable elements. In particular, the Crmar2 consensus encodes a D,D41D motif, a variant of the D,D34D catalytic domain of mariner elements. Phylogenetic analysis of the relationships of these three elements and other members of the mariner/Tc1 superfamily, based on their encoded amino acid sequences, suggests that they form a new basal subfamily of mariner elements, the rosa subfamily. BLAST analyses identified sequences from other diptera, including Drosophila melanogaster, which appear to be members of the rosa subfamily of mariner elements. Analyses of their molecular evolution suggests that Crmar2 entered the genome of C. rosa in the recent past, a consequence of horizontal transfer.     

Specificity of modified Drosophila mariner transposons in the identification of Leishmania genes

Genetic manipulation of the protozoan Leishmania has led to a better understanding of the survival and development of these pathogens within their hosts. The association of the Leishmania genome sequencing information with the ability of transposons to introduce or destroy phenotypes allows a global perspective on the role and importance of genes in cellular pathways. Herein we report the construction and testing of mariner transposable elements carrying the neomycin phosphotransferase, green fluorescent protein, or beta-glucuronidase genes as reporters for translational fusion events. We demonstrate that the expression of the reporter genes will occur only when the genes are inserted in-frame within predicted genes. Our results not only add to the mariner toolkit for gene manipulation but also strengthen the evidence that the mariner system is a reliable means for the study of gene expression in Leishmania.     

Interplasmid transposition of the mariner transposable element in non-drosophilid insects

Plasmid-based transposition assays were performed in developing embryos of the Australian sheep blowfly Lucilia cuprina and the Queensland fruit fly Bactrocera tryoni, using the mariner transposable element from Drosophila mauritiana. Transposition products were recovered that were identical in structure to those recovered from D. melanogaster. Only sequences delimited by the mariner terminal repeats were transposed and all insertions occurred at TA residues, and duplicated these. These are the hallmarks of mariner transpositions observed in the chromosomes of D. melanogaster and D. mauritiana, indicating that the plasmid-based assays are accurate indicators of mariner transposition activity. The recovery of precise transposition products from L. cuprina and B. tryoni demonstrates that mariner should be capable of producing germline transformants in these species. The results obtained from these assays suggests that they will be extremely useful in determining if mariner can transpose in other non-drosophilid insects and for investigating factors that might affect mariner transposition frequency.     

Factors affecting transposition of the Himar1 mariner transposon in vitro.

Mariner family transposable elements are widespread in animals, but their regulation is poorly understood, partly because only two are known to be functional. These are particular copies of the Dmmar1 element from Drosophila mauritiana, for example, Mos1, and the consensus sequence of the Himar1 element from the horn fly, Haematobia irritans. An in vitro transposition system was refined to investigate several parameters that influence the transposition of Himar1. Transposition products accumulated linearly over a period of 6 hr. Transposition frequency increased with temperature and was dependent on Mg2+ concentration. Transposition frequency peaked over a narrow range of transposase concentration. The decline at higher concentrations, a phenomenon observed in vivo with Mos1, supports the suggestion that mariners may be regulated in part by "overproduction inhibition." Transposition frequency decreased exponentially with increasing transposon size and was affected by the sequence of the flanking DNA of the donor site. A noticeable bias in target site usage suggests a preference for insertion into bent or bendable DNA sequences rather than any specific nucleotide sequences beyond the TA target site.     

Unexpected stability of mariner transgenes in Drosophila

A number of mariner transformation vectors based on the mauritiana subfamily of transposable elements were introduced into the genome of Drosophila melanogaster and examined for their ability to be mobilized by the mariner transposase. Simple insertion vectors were constructed from single mariner elements into which exogenous DNA ranging in size from 1.3 to 4.5 kb had been inserted; composite vectors were constructed with partial or complete duplications of mariner flanking the exogenous DNA. All of the simple insertion vectors showed levels of somatic and germline excision that were at least 100-fold lower than the baseline level of uninterrupted mariner elements. Although composite vectors with inverted duplications were unable to be mobilized at detectable frequencies, vectors with large direct duplications of mariner could be mobilized. A vector consisting of two virtually complete elements flanking exogenous DNA yielded a frequency of somatic eye-color mosaicism of approximately 10% and a frequency of germline excision of 0.04%. These values are far smaller than those observed for uninterrupted elements. The results imply that efficient mobilization of mariner in vivo requires the presence and proper spacing of sequences internal to the element as well as the inverted repeats.     

The mariner transposable element in natural populations of Drosophila simulans

In cosmopolitan species, geographical variations in copy number and/or level of transposition activity have been observed for several transposable elements (TEs). Environment, history and population structure can contribute to such variation in ways that are difficult to tease apart. For the mariner element, previous studies of the geographic variation of its somatic activity in natural populations of Drosophila simulans have shown contradictory results (latitudinal clines of divergent orientations or no apparent structure). To try and resolve these inconsistencies, we gathered all available data on the mariner somatic activity of worldwide natural populations. This includes previously published results by different groups and also new data. The correlations between the level of activity and several geoclimatic factors were tested. Although no general effect of temperature was found, a relationship with the invasion history was detected. It was also shown that recent invasive populations have a higher level of activity than the putative ancestral ones. Our results strongly suggest that variability of the mariner somatic activity among natural populations of D. simulans is mainly due to populational and historical factors probably related to the recent world colonization of this species. Indeed, this activity is correlated to the main route out of Africa (the Nile route) and the recent colonization of continents such as Australia and South America.     

The genetics and evolution of the mariner transposable element in Drosophila simulans: worldwide distribution and experimental population dynamics

We have studied both the frequency and biogeographical distribution of the transposable DNA element mariner in natural populations of Drosophila simulans and the short-term evolutionary characteristics of mariner in experimental populations. The mariner element has been identified in natural populations of D. simulans from Africa, Europe, the Middle East, Japan, Australia, several Pacific islands, North America, and South America. Only four lines out of 296 were devoid of active mariner elements, as measured by the presence of functional mariner transposase. A slight correlation was found between the latitudinal coordinate of the collection sites and the level of mariner activity in the population; this correlation became highly significant in Australia where a cline in mariner activity was observed along the eastern coast of the continent. We also observed that wild-type laboratory strains kept for several years as small populations might lose mariner activity over time. Using experimental populations, we modeled what might happen when naturally occurring populations exhibiting high and low levels of mariner activity encounter one another. We found that active mariner elements either will tend to lose their activity over time and gradually become inactive or possibly will be lost from the population; in either case, this will lead to the pattern seen in this experiment of a significant loss of mariner activity over time. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polymorphisms flanking the mariner integration sites in the rice gall midge (Orseolia oryzae Wood-Mason) genome are biotype-specific.

In an effort to study genome diversity within and between the Indian biotypes of the Asian rice gall midge, Orseolia oryzae, a major insect pest of rice, we made use of mariner transposable element integration site polymorphisms. Using degenerate primers, the design of which is based on mariner sequences, we amplified a ca. 450 bp mariner sequence from the rice gall midge. The mariner sequence showed homology with that of a mariner element isolated from the Hessian fly, Mayetiola destructor, a major dipteran pest of wheat. Southern hybridization, using this mariner fragment as a probe, revealed that the mariner elements are moderately to highly repetitive in the rice gall midge genome. Based on the sequence information of this 450-bp PCR-amplified fragment, outward-directed primers were designed and used in an inverse PCR (iPCR) to amplify the DNA flanking the conserved regions. To study the regions flanking the mariner integration sites, we employed a novel PCR-based approach: a combination of sequence specific amplification polymorphism (SSAP) and amplified fragment length polymorphism (AFLP). The outward-directed mariner-specific primer was used in combination with adapter-specific primers with 1-3 selective nucleotides at their 3' ends. The amplification products were resolved on an agarose gel, Southern-transferred onto nylon membranes, and probed with the iPCR fragment. Results revealed biotype-specific polymorphisms in the regions flanking the mariner integration sites, suggesting that mariner elements in the rice gall midge may be fixed in a biotype-specific manner. The implications of these results are discussed in the context of biotype differentiation.