Cloning and characterization of Acmar1, a mariner-like element in the asiatic honey bee,Apis cerana japonica (Hymenoptera,Apocrita)

A mariner-like element was cloned from the genome of the Asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita). The (composite) clone, named Acmar1, was 1,378 bp long, and encoded 336 amino acids corresponding to a transposase-like putative polypeptide in a single open reading frame. The D,D(34)D motif, the catalytic domain of the mariner transposase, was present, although there was a deletion of five amino acid residues within it as compared with the active transposase in Drosophila mauritiana. Nineteen-bp-long imperfect inverted terminal repeat-like sequences flanked by TA dinucleotides, the typical target site for mariner insertion, were observed. Southern blot analysis using a fragment covering two-thirds of the Acmar1 transposase coding sequence as a probe indicated the presence of multiple Acmar1-like elements in the genome. Maximum-parsimony phylogenetic analysis based on the transposase amino acid sequences of insect mariner-like elements revealed that Acmar1 is a member of the mellifera subfamily.     

Molecular characterization of a mariner-like element in the Atta sexdens rubropilosa genome

Mobile elements are widely present in eukaryotic genomes. They are repeated DNA segments that are able to move from one locus to another within the genome. They are divided into two main categories, depending on their mechanism of transposition, involving RNA (class I) or DNA (class II) molecules. The mariner-like elements are class II transposons. They encode their own transposase, which is necessary and sufficient for transposition in the absence of host factors. They are flanked by a short inverted terminal repeat and a TA dinucleotide target site, which is duplicated upon insertion. The transposase consists of two domains, an N-terminal inverted terminal repeat binding domain and a C-terminal catalytic domain. We identified a transposable element with molecular characteristics of a mariner-like element in Atta sexdens rubropilosa genome. Identification started from a PCR with degenerate primers and queen genomic DNA templates, with which it was possible to amplify a fragment with mariner transposable-element homology. Phylogenetic analysis demonstrated that this element belongs to the mauritiana subfamily of mariner-like elements and it was named Asmar1. We found that Asmar1 is homologous to a transposon described from another ant, Messor bouvieri. The predicted transposase sequence demonstrated that Asmar1 has a truncated transposase ORF. This study is part of a molecular characterization of mobile elements in the Atta spp genome. Our finding of mariner-like elements in all castes of this ant could be useful to help understand the dynamics of mariner-like element distribution in the Hymenoptera.     

A highly repetitive, mariner-like element in the genome of Hyalophora cecropia.

A highly repetitive DNA element, homologous to the mariner transposon of Drosophila mauritiana was found in the intron of the gene for cecropin A, an antibacterial peptide from the Cecropia moth. The mariner-like elements (MLE) represent a homogeneous population with a copy number of about 1000/genome. Sequencing analysis showed it to be 1255 base pairs long, including 38-base pair terminal inverted repeats. The MLE contains a defective reading frame. Nevertheless, the putative product is clearly homologous to the predicted translation product encoded by mariner. In consonance is also the fact that the inverted repeats are highly conserved between the two elements and that the overall DNA homology is 48%. Since the mariner element is present in several Drosophila species closely related to Drosophila melanogaster and since MLE is present in the lepidopteran Cecropia, a route of horizontal transfer is indicated rather than vertical transmission from a common ancestor. This suggests the possible use of mariner for the construction of an interspecies vector.     

Occurrence and abundance of a mariner-like element in freshwater and terrestrial planarians (Platyhelminthes, Tricladida) from southern Brazil

Transposable elements are DNA sequences present in all the large phylogenetic groups, both capable of changing position within the genome and constituting a significant part of eukaryotic genomes. The mariner family of transposons is one of the few which occurs in a wide variety of taxonomic groups, including freshwater planarians. Nevertheless, so far only five planarian species have been reported to carry mariner-like elements (MLEs), although several different species have been investigated. Regarding the number of copies of MLEs, Girardia tigrina is the only planarian species in which this has been evaluated, with an estimation of 8,000 copies of the element per haploid genome. Preliminary results obtained in our laboratory demonstrated that MLE is found in a large number of different species of planarians, including terrestrial. With this in mind, the aim was to evaluate the occurrence and estimate the number of MLE copies in different planarian species collected in south Brazil. Twenty-eight individuals from 15 planarian species were analyzed. By using PCR and the hybridization of nucleic acids, it was found that MLE was present in all the analyzed species, the number of copies being high, probably over 10(3) per haploid genome.     

Reduced Germline Mobility of a Mariner Vector Containing Exogenous DNA: Effect of Size or Site?

Germline mobilization of the transposable element mariner is severely inhibited by the insertion of a 4.5- to 11.9-kb fragment of exogenous DNA into a unique SacI site approximately in the middle of the 1286-bp element. In the presence of transposase driven by the germline-specific hsp26-sgs3 promoter, mobilization of the MlwB construct (containing a 11.9-kb insertion) is detected at low frequency. Analysis of a mobilized MlwB element indicated that mobilization is mediated by the mariner transposase. However, transposed MlwB elements are also defective in germline mobilization. Rare, transposase-induced germ-line excision events were also recovered for such vectors. The estimated rate of excision is <0.1% per chromosome per generation. Excision appears to be accompanied by gap repair if a suitable template is available. The data imply that the reduced mobility of mariner vectors with exogenous DNA in the SacI site results from disruption of sequences necessary for efficient mobilization. The relative stability may be a valuable property in the uses of mariner-like elements in genetic engineering of insects of economic importance.     

Identification of two mariner-like elements in the genome of the mosquito Ochlerotatus atropalpus

Two distinct mariner-like elements, Atmar-1 and Atmar-2, were isolated from the genome of the mosquito Ochlerotatus atropalpus. Full-sized Atmar-1 elements, obtained by screening a genomic library, have a 1293-bp consensus sequence with 27-bp inverted terminal repeats and a 1047-bp open reading frame (ORF) encoding the transposase. The Atmar-2 elements were amplified by polymerase chain reaction from genomic DNA and contain the central part of the transposase ORF. Individual clones of both mariner elements contain deletions, frameshifts, and stop codons. The Atmar-1 elements are present in 370-1200 copies, while the Atmar-2 elements are present in approximately 100-300 copies per haploid genome. One of the Atmar-1 elements, Atmar-1.33, could be mobilized, suggesting the presence of functional Atmar-1 elements elsewhere in the genome. Phylogenetic analysis demonstrated that Atmar-1 elements belong to the irritans subfamily and Atmar-2 elements to the cecropia subfamily of mariner elements.     

Evidence that a family of miniature inverted-repeat transposable elements (MITEs) from the Arabidopsis thaliana genome has arisen from a pogo-like DNA transposon

Sequence similarities exist between terminal inverted repeats (TIRs) of some miniature inverted-repeat transposable element (MITE) families isolated from a wide range of organisms, including plants, insects, and humans, and TIRs of DNA transposons from the pogo family. We present here evidence that one of these MITE families, previously described for Arabidopsis thaliana, is derived from a larger element encoding a putative transposase. We have named this novel class II transposon Lemi1. We show that its putative product is related to transposases of the Tc1/mariner superfamily, being closer to the pogo family. A similar truncated element was found in a tomato DNA sequence, indicating an ancient origin and/or horizontal transfer for this family of elements. These results are reminiscent of those recently reported for the human genome, where other members of the pogo family, named Tiggers, are believed to be responsible for the generation of abundant MITE-like elements in an early primate ancestor. These results further suggest that some MITE families, which are highly reiterated in plant, insect, and human genomes, could have arisen from a similar mechanism, implicating pogo-like elements.     

Survey of transposable elements from rice genomic sequences

Oryza sativa L. (domesticated rice) is a monocotyledonous plant, and its 430 Mb genome has been targeted for complete sequencing. We performed a high-resolution computer-based survey for transposable elements on 910 Kb of rice genomic DNA sequences. Both class I and II transposable elements were present, contributing 19.9% of the sequences surveyed. Class II elements greatly outnumbered class I elements (166 versus 22), although class I elements made up a greater percentage (12.2% versus 6.6%) of nucleotides surveyed. Several Mutator-like elements (MULEs) were identified, including rice elements that harbor truncated host cellular genes. MITEs (miniature inverted-repeat transposable elements) account for 71.6% of the mined transposable elements and are clearly the predominant type of transposable element in the sequences examined. Moreover, a putative Stowaway transposase has been identified based on shared sequence similarity with the mined MITEs and previously identified plant mariner-like elements (MLEs). Members of a group of novel rice elements resembling the structurally unusual members of the Basho family in Arabidopsis suggest a wide distribution of these transposons among plants. Our survey provides a preview of transposable element diversity and abundance in rice, and allows for comparison with genomes of other plant species.     

Evolution of the Transposable Element Mariner in Drosophila Species

The distribution of the transposable element mariner was examined in the genus Drosophila. Among the eight species comprising the melanogaster species subgroup, the element is present in D. mauritiana, D. simulans, D. sechellia, D. yakuba and D. teissieri, but it is absent in D. melanogaster, D. erecta and D. orena. Multiple copies of mariner were sequenced from each species in which the element occurs. The inferred phylogeny of the elements and the pattern of divergence were examined in order to evaluate whether horizontal transfer among species or stochastic loss could better account for the discontinuous distribution of the element among the species. The data suggest that the element was present in the ancestral species before the melanogaster subgroup diverged and was lost in the lineage leading to D. melanogaster and the lineage leading to D. erecta and D. orena. This inference is consistent with the finding that mariner also occurs in members of several other species subgroups within the overall melanogaster species group. Within the melanogaster species subgroup, the average divergence of mariner copies between species was lower than the coding region of the alcohol dehydrogenase (Adh) gene. However, the divergence of mariner elements within species was as great as that observed for Adh. We conclude that the relative sequence homogeneity of mariner elements within species is more likely a result of rapid amplification of a few ancestral elements than of concerted evolution. The mariner element may also have had unequal mutation rates in different lineages.     

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.     

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.     

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.     

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

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

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.     

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.     

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.     

Molecular and Functional Analysis of the Mariner Mutator Element Mos1 in Drosophila

The white-peach allele in Drosophila results from insertion of the transposable element mariner. The particular copy that is inserted in white-peach is an inactive copy referred to as the peach element. The peach element is excised at a high rate in the presence of active copies of mariner located elsewhere in the genome, and the excision of peach in somatic cells is recognized phenotypically by the occurrence of eye-color mosaicism in white-peach flies. Active mariner elements identified by their ability to induce high levels of white-peach mosaicism are denoted Mos (Mosaic) factors. We have sequenced and functionally analyzed the factor Mos1 originally identified in Drosophila mauritiana. The Mos1 element is 1286 base pairs in length, the same length as the peach element. It differs from the peach element in 11 nucleotide positions distributed throughout its length, including four amino acid replacements in the long open reading frame. Analysis of chimeric constructs between Mos1 and peach implies that functionally important differences occur in both the 5' and 3' halves of Mos1. A mariner element identical in sequence to Mos1 yields lower levels of mosaicism in transformants, implying that adjacent flanking sequences have important effects on Mos1 activity. Another mariner element, designated Ma351, isolated from a nonmosaic strain of D. mauritiana, differs from Mos1 in just three nucleotide positions. When introduced into the germline, Ma351 yields various levels of white-peach mosaicism depending on insertion site. These results imply that the activity of mariner elements is determined jointly by their own nucleotide sequences, by the effects of adjacent flanking sequences, and by longer-range position effects.     

Cloning and characterization of mariner-like elements in the soybean aphid, Aphis glycines Matsumura

Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is currently the most important insect pest of soybean (Glycine max (L.) Merr.) in the United States and causes significant economic damage worldwide, but little is known about the aphid at the molecular level. Mariner-like transposable elements (MLEs) are ubiquitous within the genomes of arthropods and various other invertebrates. In this study, we report the cloning of MLEs from the soybean aphid genome using degenerate PCR primers designed to amplify conserved regions of mariner transposases. Two of the ten sequenced clones (designated as Agmar1 and Agmar2) contained partial but continuous open reading frames, which shared high levels of homology at the protein level with other mariner transposases from insects and other taxa. Phylogenetic analysis revealed Agmar1 to group within the irritans subfamily of MLEs and Agmar2 within the mellifera subfamily. Southern blot analysis and quantitative PCR analysis indicated a low copy number for Agmar1-like elements within the soybean aphid genome. These results suggest the presence of at least two different putative mariner-like transposases encoded by the soybean aphid genome. Both Agmar1 and Agmar2 could play influential roles in the architecture of the soybean aphid genome. Transposable elements are also thought to potentially mediate resistance in insects through changes in gene amplification and mutations in coding sequences. Finally, Agmar1 and Agmar2 may represent useful genetic tools and provide insights on A. glycines adaptation.     

A rice Tc1/mariner-like element transposes in yeast

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