Testing the palindromic target site model for DNA transposon insertion using the Drosophila melanogaster P-element

Understanding the molecular mechanisms that influence transposable element target site preferences is a fundamental challenge in functional and evolutionary genomics. Large-scale transposon insertion projects provide excellent material to study target site preferences in the absence of confounding effects of post-insertion evolutionary change. Growing evidence from a wide variety of prokaryotes and eukaryotes indicates that DNA transposons recognize staggered-cut palindromic target site motifs (TSMs). Here, we use over 10 000 accurately mapped P-element insertions in the Drosophila melanogaster genome to test predictions of the staggered-cut palindromic target site model for DNA transposon insertion. We provide evidence that the P-element targets a 14-bp palindromic motif that can be identified at the primary sequence level, which predicts the local spacing, hotspots and strand orientation of P-element insertions. Intriguingly, we find that the although P-element destroys the complete 14-bp target site upon insertion, the terminal three nucleotides of the P-element inverted repeats complement and restore the original TSM, suggesting a mechanistic link between transposon target sites and their terminal inverted repeats. Finally, we discuss how the staggered-cut palindromic target site model can be used to assess the accuracy of genome mappings for annotated P-element insertions.     

New transposable elements identified as insertions in rice transposon Tnr1

Tnr1 (235 bp long) is a transposable element in rice. Polymerase chain reactions (PCRs) done with a primer(s) that hybridizes to terminal inverted repeat sequences (TIRs) of Tnr1 detected new Tnr1 members with one or two insertions in rice genomes. Six identified insertion sequences (Tnr4, Tnr5, Tnr11, Tnr12, Tnr13 and RIRE9) did not have extensive homology to known transposable elements, rather they had structural features characteristic of transposable elements. Tnr4 (1767 bp long) had imperfect 64-bp TIRs and appeared to generate duplication of a 9-bp sequence at the target site. However, the TIR sequences were not homologous to those of known transposable elements, indicative that Tnr4 is a new transposable element. Tnr5 (209 bp long) had imperfect 46-bp TIRs and appeared to generate duplication of sequence TTA like that of some elements of the Tourist family. Tnr11 (811 bp long) had 73-bp TIRs with significant homology to those of Tnr1 and Stowaway and appeared to generate duplication of sequence TA, indicative that Tnr11 is a transposable element of the Tnr1/Stowaway family. Tnr12 (2426 bp long) carried perfect 9-bp TIRs, which began with 5'-CACTA- -3' from both ends and appeared to generate duplication of a 3-bp target sequence, indicative that Tnr12 is a transposable element of the En/Spm family. Tnr13 (347 bp long) had 31-bp TIRs and appeared to generate duplication of an 8-bp target sequence. Two sequences, one the transposon-like element Crackle, had partial homology in the Tnr13 ends. All five insertions appear to be defective elements derived from autonomous ones encoding the transposase gene. All had characteristic tandem repeat sequences which may be recognized by transposase. The sixth insertion sequence, named RIRE9 (3852 bp long), which begins with 5'-TG- -3' and ends with 5'- -CA-3', appeared to generate duplication of a 5-bp target sequence. These and other structural features indicate that this insertion is a solo LTR (long terminal repeat) of a retrotransposon. The transposable elements described above could be identified as insertions into Tnr1, which do not deleteriously affect the growth of rice cells.     

Vege and Mar: two novel hAT MITE families from Drosophila willistoni

Two novel families of miniature inverted repeat transposable elements (MITEs), Vege and Mar, are described from Drosophila willistoni. Based on their structures, both element families are hypothesized to belong to the hAT superfamily of transposable elements. Both elements have perfect, inverted terminal repeats and 8-bp target site duplications and were found to have inserted within fixed copies of nonautonomous P elements. Vege is present in all studied D. willistoni populations and appears to have a relatively low copy number. Mar was identified in only a single D. willistoni population, and its copy number is presently unknown. Although MITEs occupy relatively large proportions of the genomes of a broad range of organisms, this may be their first unambiguous identification in any species of the genus Drosophila.     

Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.     

Tm1: a mutator/foldback transposable element family in root-knot nematodes

Three closely related parthenogenetic species of root-knot nematodes, collectively termed the Meloidogyne incognita-group, are economically significant pathogens of diverse crop species. Remarkably, these asexual root-knot nematodes are capable of acquiring heritable changes in virulence even though they lack sexual reproduction and meiotic recombination. Characterization of a near isogenic pair of M. javanica strains differing in response to tomato with the nematode resistance gene Mi-1 showed that the virulent strain carried a deletion spanning a gene called Cg-1. Herein, we present evidence that the Cg-1 gene lies within a member of a novel transposable element family (Tm1; Transposon in Meloidogyne-1). This element family is defined by composite terminal inverted repeats of variable lengths similar to those of Foldback (FB) transposable elements and by 9 bp target site duplications. In M. incognita, Tm1 elements can be classified into three general groups: 1) histone-hairpin motif elements; 2) MITE-like elements; 3) elements encoding a putative transposase. The predicted transposase shows highest similarity to gene products encoded by aphids and mosquitoes and resembles those of the Phantom subclass of the Mutator transposon superfamily. Interestingly, the meiotic, sexually-reproducing root-knot nematode species M. hapla has Tm1 elements with similar inverted repeat termini, but lacks elements with histone hairpin motifs and contains no elements encoding an intact transposase. These Tm1 elements may have impacts on root-knot nematode genomes and contribute to genetic diversity of the asexual species.     

Transposon excision from an atypical site: a mechanism of evolution of novel transposable elements

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3' to 5' trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations.     

Mechanisms regulating the copy numbers of six LTR retrotransposons in the genome of Drosophila melanogaster

There has been debate over the mechanisms that control the copy number of transposable elements in the genome of Drosophila melanogaster. Target sites in D. melanogaster populations are occupied at low frequencies, suggesting that there is some form of selection acting against transposable elements. Three main theories have been proposed to explain how selection acts against transposable elements: insertions of a copy of a transposable element are selected against; chromosomal rearrangements caused by ectopic exchange between element copies are selected against; or the process of transposition itself is selected against. The three theories give different predictions for the pattern of transposable element insertions in the chromosomes of D. melanogaster. We analysed the abundance of six LTR (long terminal repeat) retrotransposons on the X and fourth chromosomes of multiple strains of D. melanogaster, which we compare with the predictions of each theory. The data suggest that no one theory can account for the insertion patterns of all six retrotransposons. Comparing our results with earlier work using these transposable element families, we find a significant correlation between studies in the particular model of copy number regulation supported by the proportion of elements on the X for the different transposable element families. This suggests that different retrotransposon families are regulated by different mechanisms.     

A new transposable element in Chironomus thummi

Summary A 1.7 kb long transposable element called TECth1 was found in the 3 flanking region of aChironomus thummi Balbiani ring gene. As shown by sequence comparison with a second copy, TECthl is characterized by a perfect terminal inverted repeat of 17 by flanked by a duplicated target site of 8 bp, four internal imperfect inverted repeats of 17 to 26 by and terminal regions of about 0.25 kb with a high number of short direct repeats of the consensus sequence ACTTT or permutated and mutated forms such as TTTAC or ACTAT. The terminal inverted repeats and the 8 by target site duplication are reminiscent ofDrosophila P and hobo elements but no long open reading frame starting with ATG is present, suggesting that the two TECthl copies studied represent deletion derivatives of a longer element coding for its own transposase. In situ hybridization revealed about 75 labelled sites distributed over all chromosomes with the Balbiani ring locus most strongly labelled. Fifty percent of the sites are specific for a given individual, and these variable sites are often heterozygous for the element.     

The Tc2 transposon of Caenorhabditis elegans has the structure of a self-regulated element.

We have analyzed the sequence of the Tc2 transposon of the nematode Caenorhabditis elegans. The Tc2 element is 2,074 bp in length and has perfect inverted terminal repeats of 24 bp. The structure of this element suggests that it may have the capacity to code for a transposase protein and/or for regulatory functions. Three large reading frames on one strand exhibit nonrandom codon usage and may represent exons. The first open coding region is preceded by a potential CAAT box, TATA box, and consensus heat shock sequence. In addition to its inverted terminal repeats, Tc2 has an unusual structural feature: subterminal degenerate direct repeats that are arranged in an irregular overlapping pattern. We have also examined the insertion sites of two Tc2 elements previously identified as the cause of restriction fragment length polymorphisms. Both insertions generated a target site duplication of 2 bp. One element had inserted inside the inverted terminal repeat of another transposon, splitting it into two unequal parts.     

DNA sequence requirements for hobo transposable element transposition in Drosophila melanogaster

We have conducted a structure and functional analysis of the hobo transposable element of Drosophila melanogaster. A minimum of 141 bp of the left (L) end and 65 bp of the right (R) end of the hobo were shown to contain sequences sufficient for transposition. Both ends of hobo contain multiple copies of the motifs GGGTG and GTGGC and we show that the frequency of hobo transposition increases as a function of the copy number of these motifs. The R end of hobo contains a unique 12 bp internal inverted repeat that is identical to the hobo terminal inverted repeats. We show that this internal inverted repeat suppresses transposition activity in a hobo element containing an intact L end and only 475 bp of the R end. In addition to establishing cis-sequences requirements for transposition, we analyzed trans-sequence effects of the hobo transposase. We show a hobo transposase lacking the first 49 amino acids catalyzed hobo transposition at a higher frequency than the full-length transposase suggesting that, similar to the related Ac transposase, residues at the amino end of the transposase reduce transposition. Finally, we compared target site sequences of hobo with those of the related Hermes element and found both transposons have strong preferences for the same insertion sites.     

Nonautonomous inverted repeat Alien transposable elements are associated with genes of both monocotyledonous and dicotyledonous plants

Alien are highly repeated plant transposable elements characterized by their small size (approx. 400 bp), high A + T content, target site specificity, potential to form stable secondary structures and possession of a conserved 28-bp terminal inverted repeat (TIR). Besides the TIR, they contain subterminal inverted repeat motifs (SIRM), as well as the 5′-CATGCAT domain which has been reported to be a cis-acting regulatory element of gene expression in some plant species. Although they were first identified in the intron of the bell pepper (Capsicum annuum) Sn-2 gene and in the promoter region of the potato starch phosphorylase-encoding gene, Alien arranged in tandem are present in the promoter of patatin class-II genes. PCR on the bell pepper genomic DNA using the Alien TIR consensus sequence as primer yielded DNA fragments of nearly 400 bp. These fragments have characteristics of transposable elements and contain numerous motifs reminiscent of Alien elements. Importantly, PCR on genomic DNA extracts from various monocotyledonous and dicotyledonous plants using the TIR consensus sequence as primer and subsequent hybridization with different Alien probes revealed that these elements are ubiquitously present and highly repeated in the genomes of higher plants.     

MAK,a computational tool kit for automated MITE analysis

Miniature inverted repeat transposable elements (MITEs) are ubiquitous and numerous in higher eukaryotic genomes. Analysis of MITE families is laborious and time consuming, especially when multiple MITE families are involved in the study. Based on the structural characteristics of MITEs and genetic principles for transposable elements (TEs), we have developed a computational tool kit named MITE analysis kit (MAK) to automate the processes (http://perl.idmb.tamu.edu/mak.htm). In addition to its ability to routinely retrieve family member sequences and to report the positions of these elements relative to the closest neighboring genes, MAK is a powerful tool for revealing anchor elements that link MITE families to known transposable element families. Implementation of the MAK is described, as are genetic principles and algorithms used in its derivation. Test runs of the programs for several MITE families yielded anchor sequences that retain TIRs and coding regions reminiscent of transposases. These anchor sequences are consistent with previously reported putative autonomous elements for these MITE families. Furthermore, analysis of two MITE families with no known links to any transposon family revealed two novel transposon families, namely Math and Kid, belonging to the IS5/Harbinger/PIF superfamily.     

Assembly of the mariner Mos1 synaptic complex

The mobility of transposable elements via a cut-and-paste mechanism depends on the elaboration of a nucleoprotein complex known as the synaptic complex. We show here that the Mos1 synaptic complex consists of the two inverted terminal repeats of the element brought together by a transposase tetramer and is designated paired-end complex 2 (PEC2). The assembly of PEC2 requires the formation of a simpler complex, containing one terminal repeat and two transposase molecules and designated single-end complex 2 (SEC2). In light of the formation of SEC2 and PEC2, we demonstrate the presence of two binding sites for the transposase within a single terminal repeat. We have found that the sequence of the Mos1 inverted terminal repeats contains overlapping palindromic and mirror motifs, which could account for the binding of two transposase molecules "side by side" on the same inverted terminal repeat. We provide data indicating that the Mos1 transposase dimer is formed within a single terminal repeat through a cooperative pathway. Finally, the concept of a tetrameric synaptic complex may simply account for the inability of a single mariner transposase molecule to interact at the same time with two kinds of DNA: the inverted repeat and the target DNA.     

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.     

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.     

Small mobile sequences in bacteria display diverse structure/function motifs

Small repeat sequences in bacterial genomes, which represent non-autonomous mobile elements, have close similarities to archaeon and eukaryotic miniature inverted repeat transposable elements. These repeat elements are found in both intergenic and intragenic chromosomal regions, and contain an array of diverse motifs. These can include DNA sequences containing an integration host factor binding site and a proposed DNA methyltransferase recognition site, transcribed RNA secondary structural motifs, which are involved in mRNA regulation, and translated open reading frames found fused to other open reading frames. Some bacterial mobile element fusions are in evolutionarily conserved protein and RNA genes. Others might represent or lead to creation of new protein genes. Here we review the remarkable properties of these small bacterial mobile elements in the context of possible beneficial roles resulting from random insertions into the genome.