Transposition behavior of nonautonomous a <Emphasis Type="Italic">hAT</Emphasis> superfamily transposon <Emphasis Type="Italic">nDart</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0–13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.     

Identification of a high frequency transposon induced by tissue culture,nDaiZ, a member of the hAT family in rice

Recent completion of rice genome sequencing has revealed that more than 40% of its genome consists of repetitive sequences, and most of them are related to inactive transposable elements. In the present study, a transposable element, nDaiZ0, which is induced by tissue culture with high frequency, was identified by sequence analysis of an allelic line of the golden hull and internode 2 (gh2) mutant, which was integrated into the forth exon of GH2. The 528-bp nDaiZ0 has 14-bp terminal inverted repeats (TIRs), and generates an 8-bp duplication of its target sites (TSD) during its mobilization. nDaiZs are non-autonomous transposons and have no coding capacity. Bioinformatics analysis and southern blot hybridization showed that at least 16 copies of nDaiZ elements exist in the japonica cultivar Nipponbare genome and 11 copies in the indica cultivar 93-11 genome. During tissue culture, only one copy, nDaiZ9, located on chromosome 5 in the genome of Nipponbare can be activated with its transposable frequency reaching 30%. However, nDaiZ9 was not present in the 93-11 genome. The larger elements, DaiZs, were further identified by database searching using nDaiZ0 as a query because they share similar TIRs and subterminal sequences. DaiZ can also generate an 8-bp TSD. DaiZ elements contain a conserved region with a high similarity to the hAT dimerization motif, suggesting that the nDaiZ–DaiZ transposon system probably belongs to the hAT superfamily of class II transposons. Phylogenetic analysis indicated that it is a new type of plant hAT-like transposon. Although nDaiZ is activated by tissue culture, the high transposable frequency indicates that it could become a useful gene tagging system for rice functional genomic studies. In addition, the mechanism of the high transposable ability of nDaiZ9 is discussed.     

A candidate autonomous version of the wheat MITE <Emphasis Type="Italic">Hikkoshi</Emphasis> is present in the rice genome

A miniature inverted-repeat transposable element (MITE), designated as Hikkoshi, was previously identified in the null Wx-A1 allele of Turkish bread wheat lines. This MITE is 165 bp in size and has 12-bp terminal inverted repeats (TIRs) flanked by 8-bp target site duplications (TSDs). Southern and PCR analyses demonstrated the presence of multiple copies of Hikkoshi in the wheat genome. Database searches indicated that Hikkoshi MITEs are also present in barley, rice and maize. A 3.4-kb element that has Hikkoshi-like TIRs flanked by 8-bp TSDs has now been identified in the rice genome. This element shows high similarity to the 5 subterminal region of the wheat Hikkoshi MITE and contains a transposase (TPase) coding region. The TPase has two conserved domains, ZnF_TTF and hATC, and its amino acid sequence shows a high degree of homology to TPases encoded by Tip100 transposable elements belonging to the hAT superfamily. We designated the 3.4-kb element as OsHikkoshi. Several wheat clones deposited in EST databases showed sequence similarity to the TPase ORF of OsHikkoshi. The sequence information from the TPase of OsHikkoshi will thus be useful in isolating the autonomous element of the Hikkoshi system from wheat.     

<Emphasis Type="Italic">hATpin</Emphasis>, a Family of MITE-like <Emphasis Type="Italic">hAT</Emphasis> Mobile Elements Conserved in Diverse Plant Species that Forms Highly Stable Secondary Structures

We identified a 178 bp mobile DNA element in lettuce with characteristic CGAGC/GCTCG repeats in the subterminal regions. This element has terminal inverted repeats and 8-bp target site duplications typical of the hAT superfamily of class II mobile elements, but its small size and potential to form a single-stranded stable hairpin-like secondary structure suggest that it is related to MITE elements. In silico searches for related elements identified 252 plant sequences with 8-bp target site duplications and sequence similarity in their terminal and subterminal regions. Some of these sequences were predicted to encode transposases and may be autonomous elements; these constituted a separate clade within the phylogram of hAT transposases. We demonstrate that the CGAGC/GCTCG pentamer maximizes the hairpin stability compared to any other pentamer with the same C + G content, and the secondary structures of these elements are more stable than for most MITEs. We named these elements collectively as hATpin elements because of the hAT similarity and their hairpin structures. The nearly complete rice genome sequence and the highly advanced genome annotation allowed us to localize most rice elements and to deduce insertion preferences. hATpin elements are distributed on all chromosomes, but with significant bias for chromosomes 1 and 10 and in regions of moderate gene density. This family of class II mobile elements is found primarily in monocot species, but is also present in dicot species. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 by in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fott, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tct. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.     

Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.)

Members of a novel Master family of class II transposons were identified in the carrot genome. Two elements, 2.5 kb long DcMaster1 and 4.4 kb long DcMaster-a, are characterized by 22 bp imperfect terminal inverted repeats and by 3 bp target site duplications. GenBank search revealed that related elements are also present in Medicago truncatula, including a 5.1 kb element MtMaster-a. Both DcMaster-a and MtMaster-a contain open reading frames encoding for putative transposases with the complete DDE domain typical for plant class II transposable elements belonging to PIF/Harbinger superfamily, where the Master elements form a distinct group. Less than 10 copies of the DcMaster element containing the DDE domain are present in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in 8 of 14 identified insertion sites. PCR amplification of carrot genomic DNA using a primer complementary to TIRs of DcMaster gave products <400 bp in size. We speculate that these may all represent a MITE-like family of transposable elements that we named Krak, present in the carrot genome in at least 3,600 copies. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers DQ250792 to DQ250807 and DQ353734 to DQ353752.     

A hAT superfamily transposase recruited by the cereal grass genome

Transposable elements are ubiquitous genomic parasites with an ancient history of coexistence with their hosts. A few cases have emerged recently where these genetic elements have been recruited for normal function in the host organism. We have identified an expressed hobo/Ac/Tam (hAT) family transposase-like gene in cereal grasses which appears to represent such a case. This gene, which we have called gary, is found in one or two copies in barley, two diverged copies in rice and two very similar copies in hexaploid wheat. No gary homologues are found in Arabidopsis. In all three cereal species, an apparently complete 2.5 kb transposase-like open reading frame is present and nucleotide substitution data show evidence for positive selection, yet the predicted gary protein is probably not an active transposase, as judged by the absence of key amino acids required for transposase function. Gary is expressed in wheat and barley spikes and gary cDNA sequences are also found in rice, oat, rye, maize, sorghum and sugarcane. The short inverted terminal repeats, flanked by an eight-nucleotide host sequence duplication, which are characteristic of a hAT transposon are absent. Genetic mapping in barley shows that gary is located on the distal end of the long arm of chromosome 2H. Wheat homologues of gary map to the same approximate location on the wheat group 2 chromosomes by physical bin-mapping and the more closely related of the two rice garys maps to the syntenic location near the bottom of rice chromosome 4. These data suggest that gary has resided in a single genomic location for at least 60 Myr and has lost the ability to transpose, yet expresses a transposase-related protein that is being conserved under host selection. We propose that the gary transposase-like gene has been recruited by the cereal grasses for an unknown function.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Characterization of Imcrop, a Mutator-like MITE family in the rice genome

Sequence comparisons of ammonium transporter 1?C2 genes (OsAMT1-2) in different rice accessions revealed a MITE insertion in the upstream region of the gene. The 391-bp MITE, classified as a Mutator superfamily member and named Imcrop, included terminal inverted repeat (TIR) and 9-bp target site duplication (TSD) sequences. We identified 151 Imcrop elements dispersed on 12 chromosomes of the japonica reference genome. Of these, 12.6% were found in genic regions and 33.1% were located within 1.5 kb of annotated rice genes. We constructed comparative insertion maps with 111 and 102 intact Imcrop elements in the japonica and indica reference genomes, respectively. The Imcrop elements showed relatively even distribution across all chromosomes although their frequency was higher on chromosomes 1, 3, and 4 in both genomes. Seventy seven Imcrop elements were detected in both subspecies, whereas 34 and 25 insertions were found only in the japonica or indica genome, respectively. We compared insertion polymorphisms of 19 Imcrop elements found inside genes in 48 Korean rice cultivars, consisting of 42 japonica and six Tongil-types (indica-japonica cross). Thirteen insertions were common to all cultivars indicating these elements were present before indica-japonica divergence. The six other elements showed insertion polymorphisms among accessions, showing their recent insertion history or no critical positive effect of their insertion on the rice genome.     

Transposition and target preferences of an active nonautonomous DNA transposon nDart1 and its relatives belonging to the hAT superfamily in rice

The nonautonomous nDart1 element in the hAT superfamily is one of a few active DNA transposons in rice. Its transposition can be induced by crossing with a line containing an active autonomous element, aDart1, and stabilized by segregating aDart1. No somaclonal variation should occur in nDart1-promoted gene tagging because no tissue culture is involved in nDart1 activation. By transposon display analysis, we examined the activities of nDart1-related elements in the selfed progeny of a mutable virescent pyl-v plant containing aDart1. Although various nDart1-related elements are present in the rice genome, only nDart1-3 subgroup elements, nDart1-0 and nDart1-3 in particular, were found to be transposed frequently and integrated into various sites almost all over the genome, and a fraction of the transposed elements were found to be transmitted to the next generation. More than half of the newly integrated elements were identified as nDart1-0. Analysis of the newly inserted sites revealed that the nDart1-3 subgroup elements were predominantly integrated into single-copy regions. More than 60% of the transposed elements were inserted into the genic regions that comprise putative coding regions and their 0.5-kb flanking segments, and approximately two-thirds of them were within the 0.5-kb area in front of the putative initiation codons, i.e., promoter-proximal genic regions. These characteristic features of nDart1-3 subgroup elements seem to be suitable for developing an efficient and somaclonal variation-free gene tagging system for rice functional genomics.     

<Emphasis Type="Italic">Anaconda</Emphasis>, a new class of transposon belonging to the <Emphasis Type="Italic">Mu</Emphasis> superfamily,has diversified by acquiring host genes during rice evolution

A new type of transposon, named Anaconda (Anac) has been found in rice (Oryza sativa). In this paper, we demonstrate that Anaconda elements have diversified by acquisition of host cellular genes, amplification of the elements, and substitution and deletion of short segments. We identified four Anaconda elements in studies of rice alternative oxidase (AOX) genes, and subsequently isolated an additional 23 elements based on the identity of their terminal inverted repeats (TIRs). The Anaconda elements have long TIRs (114–458 bp). They also have direct repeats of 9 or 10 bp in their flanking regions that are thought to have been generated upon transposition. These structural features reveal that the Anaconda elements belong to the Mu superfamily. The most prominent feature of the Anaconda elements is the high frequency with which they have acquired host cellular genes. Of the 27 elements found here, 19 appear to have sequences presumably derived from rice genes, for example, the genes for AOX1c (four elements), cytochrome P450 (five elements), l-asparaginase (five elements), and PCF8 (two elements). Four elements, AnacA1–A4, have both the AOX1c and P450 genes. One element, AnacB14, involves a gene similar to mudrA of maize MuDR. Database analyses revealed that the loci of 26 of the 27 Anaconda elements in the subspecies japonica are the same as those in the subspecies indica. This suggests that these elements were incorporated before the divergence of these two subspecies.     

The generation of Mutator transposable element subfamilies in maize

The mobile DNAs of the Mutator system of maize (Zea mays) are exceptional both in structure and diversity. So far, six subfamilies of Mu elements have been discovered; all Mu elements share highly conserved terminal inverted repeats (TIRs), but each sub-family is defined by internal sequences that are apparently unrelated to the internal sequences of any other Mu subfamily. The Mu1/Mu2 subfamily of elements was created by the acquisition of a portion of a standard maize gene (termed MRS-A) within two Mu TIRs. Beside the unusually long (185–359 bp) and diverse TIRs found on all of these elements, other direct and inverted repeats are often found either within the central portion of a Mu element or within a TIR.Our computer analyses have shown that sequence duplications (mostly short direct repeats interrupted by a few base pairs) are common in non-autonomous members of the Mutator, Ac/Ds, and Spm(En) systems. These duplications are often tightly associated with the element-internal end of the TIRs. Comparisons of Mu element sequences have indicated that they share more terminal components than previously reported; all subfamilies have at least the most terminal 215 bp, at one end or the other, of the 359-bp Mu5 TIR. These data suggest that many Mu element subfamilies were generated from a parental element that had termini like those of Mu5. With the Mu5 TIRs as a standard, it was possible to determine that elements like Mu4 could have had their unusual TIRs created through a three-step process involving (1) addition of sequences to interrupt one TIR, (2) formation of a stem-loop structure by one strand of the element, and (3) a subsequent DNA repair/gene conversion event that duplicated the insertion(s) within the other TIR. A similar repair/conversion extending from a TIR stem into loop DNA could explain the additional inverted repeat sequences added to the internal ends of the Mu4 and Mu7 TIRs. This same basic mechanism was found to be capable of generating new Mu element subfamilies. After endonucleolytic attack of the loop within the stem-loop structure, repair/conversion of the gap could occur as an intermolecular event to generate novel internal sequences and, therefore, a new Mu element subfamily. Evidence supporting and expanding this model of new Mu element subfamily creation was identified in the sequence of MRS-A.     

Maize Mu transposons are targeted to the 5' untranslated region of the gl8 gene and sequences flanking Mu target-site duplications exhibit nonrandom nucleotide composition throughout the genome

The widespread use of the maize Mutator (Mu) system to generate mutants exploits the preference of Mu transposons to insert into genic regions. However, little is known about the specificity of Mu insertions within genes. Analysis of 79 independently isolated Mu-induced alleles at the gl8 locus established that at least 75 contain Mu insertions. Analysis of the terminal inverted repeats (TIRs) of the inserted transposons defined three new Mu transposons: Mu10, Mu 11, and Mu12. A large percentage (>80%) of the insertions are located in the 5' untranslated region (UTR) of the gl8 gene. Ten positions within the 5' UTR experienced multiple independent Mu insertions. Analyses of the nucleotide composition of the 9-bp TSD and the sequences directly flanking the TSD reveals that the nucleotide composition of Mu insertion sites differs dramatically from that of random DNA. In particular, the frequencies at which C's and G's are observed at positions -2 and +2 (relative to the TSD) are substantially higher than expected. Insertion sites of 315 RescueMu insertions displayed the same nonrandom nucleotide composition observed for the gl8-Mu alleles. Hence, this study provides strong evidence for the involvement of sequences flanking the TSD in Mu insertion-site selection.     

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 by in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fott, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tct. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.     

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.     

Structural and sequence diversity of the transposon Galileo in the Drosophila willistoni genome

Background

Galileo is one of three members of the P superfamily of DNA transposons. It was originally discovered in Drosophila buzzatii, in which three segregating chromosomal inversions were shown to have been generated by ectopic recombination between Galileo copies. Subsequently, Galileo was identified in six of 12 sequenced Drosophila genomes, indicating its widespread distribution within this genus. Galileo is strikingly abundant in Drosophila willistoni, a neotropical species that is highly polymorphic for chromosomal inversions, suggesting a role for this transposon in the evolution of its genome.

Results

We carried out a detailed characterization of all Galileo copies present in the D. willistoni genome. A total of 191 copies, including 133 with two terminal inverted repeats (TIRs), were classified according to structure in six groups. The TIRs exhibited remarkable variation in their length and structure compared to the most complete copy. Three copies showed extended TIRs due to internal tandem repeats, the insertion of other transposable elements (TEs), or the incorporation of non-TIR sequences into the TIRs. Phylogenetic analyses of the transposase (TPase)-encoding and TIR segments yielded two divergent clades, which we termed Galileo subfamilies V and W. Target-site duplications (TSDs) in D. willistoni Galileo copies were 7- or 8-bp in length, with the consensus sequence GTATTAC. Analysis of the region around the TSDs revealed a target site motif (TSM) with a 15-bp palindrome that may give rise to a stem-loop secondary structure.

Conclusions

There is a remarkable abundance and diversity of Galileo copies in the D. willistoni genome, although no functional copies were found. The TIRs in particular have a dynamic structure and extend in different ways, but their ends (required for transposition) are more conserved than the rest of the element. The D. willistoni genome harbors two Galileo subfamilies (V and W) that diverged ~9 million years ago and may have descended from an ancestral element in the genome. Galileo shows a significant insertion preference for a 15-bp palindromic TSM.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-792) contains supplementary material, which is available to authorized users.     

Does the Upstream Region Possessing MULE-Like Sequence in Rice Upregulate PsbS1 Gene Expression?

The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type.