BACKGROUND
traditionally, transposable elements  have been classified as either class i or class ii  <cit> .
class i, or retroelements, transpose through an rna intermediate.
this group includes both long-terminal repeat  transposons and non-long-terminal repeat retrotransposons  <cit> .
class ii elements transpose via dna, include inverted repeats  <cit> , and often leave a "footprint" after excision  <cit> .
the two classes of transposable elements share at least two features: first, both duplicate host sequences during integration into the host genome, and second, the 3' end of the insertion is either a duplicate of the 5' end or a poly  tail  <cit> .
in  <dig>  kapitonov and jurka identified a new group of eukarotyic dna tes in eukaryotes that they named helitrons  <cit> .
helitrons are dna elements that are proposed to move via a rolling circle replication mechanism similar to that of some prokaryotic transposable elements  <cit> .
they are unique in that they do not duplicate host insertion sites and do not contain terminal repeats.
they can also be quite large, up to  <dig> kb or more  <cit> .
however, helitrons do have two conserved sequence features.
first, they tend to insert between adenine and thymidine residues.
second, they have conserved ends consisting of tc on the 5' end and ctrr on the 3' end, often with a palindromic sequence of 16– <dig> bp near the 3' terminus.
because they do not replicate by the "cut and paste" method of most class ii elements, helitrons were initially difficult to classify and were considered their own class.
they have now been assigned as a separate subclass of class ii tes  <cit> .
non-autonomous helitrons are common in eukaryotic genomes, representing ~2–3% of genomes in species as diverse as arabidopsis thaliana , caenorabditis elegans and bats   <cit> , but they are arguably most interesting in maize , where they contribute to substantial differences in gene order and content among individuals  <cit> .
for example, the bronze region features extensive sequence non-homologies among maize inbred lines  <cit> , in large part due to non-autonomous helitron insertions.
remarkably, much of this non-homology represents expressed gene fragments that have been captured within non-autonomous elements.
gene capture appears to be both widespread and frequent.
for example, just four helitrons were found to contain fragments that originated from at least seven genes  <cit> .
moreover, overgo hybridization suggests that as many as  <dig>  genes or gene fragments may be unshared between inbred lines, perhaps due to helitron activity  <cit> .
the presence of expressed gene fragments within helitrons highlights their potential to contribute to functional, as well as structural, diversity within plant genomes  <cit> .
the observations in maize provide a puzzling contrast to observations in arabidopsis.
arabidopsis contains > <dig> non-autonomous helitrons, many of which were initially catalogued as basho elements  <cit> .
like helitrons in maize, many a. thaliana elements contain gene fragments.
unlike maize, however, these fragments appear to represent only a small number of gene capture events  <cit> .
this contrast between maize and arabidopsis raises a number of important questions about the evolution of helitrons and their role in gene capture.
first, are the small numbers of maize helitrons characterized thus far typical with respect to the extent of gene capture, or could the apparent differences between species be due to the small number of helitrons isolated from maize thus far?
second, if one examines a third experimental system, like rice , do maize or arabidopsis helitrons appear to be more typical with respect to gene capture?
third, if there are real differences in gene capture between maize and other plant species, can one glean any clues as to the mechanistic or evolutionary factors that contribute to these differences?
RESULTS
helitron identification and polymorphism in maize
we used a blast-based approach to identify helitrons from maize genomic data .
our search began with a training set of  <dig> maize 'seed' elements from the literature  <cit> , from which we isolated both 5' and 3' ends as blast queries.
these queries were  <dig> bp in length, but generally yielded ~ <dig> bp matches corresponding to helitron ends .
this approach was applied iteratively, accruing additional blast queries, until no more ends were detected .
after careful consideration of redundancy in genbank entries, we produced a curated set of  <dig> predicted maize helitrons, with a mean length of  <dig> base pairs and a range from  <dig> bp to  <dig> bp.
we combined our predicted helitrons with the  <dig> query sequences to produces a total set of  <dig> maize helitrons .
before drawing conclusions from our predicted set of maize helitrons, it is important to examine the properties of the predicted elements.
we first examined the 3' and 5' ends for features characteristic of helitrons .
to date, maize helitrons have had 5' ends marked by insertion next to a flanking 'a' and a beginning sequence of 'tct', followed by a  <dig> bp pattern without a strong consensus  <cit> .
du et al.
<cit>  recently used this  <dig> bp pattern, including sequence ambiguities, to identify potential 5' ends of helitrons in maize genomic sequence.
our collection of  <dig> putative elements perfectly reflect the search pattern of du et al.
, but also clearly indicate a lack of strong consensus nucleotides at residues  <dig> to  <dig> .
similarly, our sample of predicted helitrons reflect the recognized preference for helitrons to terminate in 'ctagt', and the 3' ~ <dig> bp motif also match the maize consensus  <cit> .
although our search began with a seed set that reflected particular properties, there is no guarantee that our blast strategy would return strong signals in the particular residues that have previously been defined as typical of helitrons.
moreover, these patterns are qualitatively indistinguishable when the  <dig> 'seed' helitrons are removed from consideration .
in addition to 5' and 3' motifs, our results clearly indicate the lack of consensus nucleotides in flanking genomic sequences, except for the strong  bias toward inserting downstream from an 'a' .
because maize helitrons were first identified by comparisons of sequence data between maize inbred lines  <cit> , we next assessed polymorphism among maize individuals.
we attempted to survey our  <dig> predicted helitrons with a pcr assay of eight maize inbred lines .
we were unable to amplify single bands consistently for eight of the predicted helitrons, and so could not unambiguously assess polymorphism for these presumptive helitrons.
another eight were fixed in the panel of eight maize inbred lines, and polymorphism could not be verified in a broader germplasm set, either due to a lack of polymorphism or poor amplification properties.
however, five helitrons were polymorphic in the sample of inbred lines.
thus,  <dig> of our  <dig> predicted helitrons  were confirmed to be polymorphic.
we next used the 5' and 3' ends from our panel of  <dig> putative maize helitrons to build profile hmm models  and used the models to scan all maize genomic sequences in genbank.
we found that the recall rate, which is a measure of sensitivity, was very low , indicating that the hmm could not effectively model the 5' and 3' maize motifs.
the failure of the hmm approach may indicate that maize harbors a broad diversity of helitron subfamilies with widely variable 3' and 5' ends .
helitron identification and polymorphism in rice
while conducting our blast-based search for maize helitrons in genbank, we also discovered  <dig> putative helitrons in genomic rice sequences.
mirroring our approach for maize, we used the 5' and 3' ends from this panel of putative rice helitrons to build profile hmms and used the models to scan two separate sources: genomic sequences of indica and japonica rice.
in contrast to the hmm application to maize, the rice hmms achieved a much higher recall rate, suggesting that rice helitrons may share extensive sequence similarity.
the hmm models identified  <dig> putative helitron elements in the japonica rice genome , and  <dig> elements in the indica rice genome.
to determine whether we would expect to identify  <dig> elements based on chance alone, we constructed randomized rice genomes with the same length and compositional properties of japonica rice  and reapplied the hmm.
in five randomized genomes, the hmm failed to identify a single helitron, thus illustrating that our findings are not expected due to random hits to a genomic sequence of similar length and composition.
because the helitron components from the rice genomes were similar and because the japonica genome is better annotated, we focused on the set of japonica elements for description.
the mean length of predicted japonica helitrons was  <dig> bp, with 97% of these under  <dig>  kb in length and at least two of the longest  containing internal retrotransposons.
none of the putative helitrons appear to be autonomous, based both on length and on blastn comparisons to putative helicase genes .
we examined the 5' and 3' ends of predicted rice helitrons, just as we examined them in maize .
the rice helitrons generally yield stronger consensus motifs, with nucleotide biases extending for ~ <dig> bp on the 5' end and  <dig> bp on the 3' end.
nonetheless, there are shared features of the motifs between maize and rice, particularly the 5' 'tct' and the 3' 'ctagt'.
we also examined the regions flanking rice helitrons.
like maize, the helitrons exhibited a preference for a 5' flanking 'a' nucleotide.
the 3' flanking sequences were markedly a+t rich, which is also a preference in maize but less pronounced .
the strong motif pattern in rice terminal regions suggested that we had uncovered a well-conserved group of helitrons.
to pursue this suspicion, we separated the putative helitrons into single-linkage clusters, yielding one particularly large cluster of  <dig> elements for which we constructed a neighbor-joining tree .
with this tree, we were able to infer approximate times of element insertion.
using the terminal branch lengths  in the phylogeny as a proxy for time, and assuming a molecular clock with a nucleotide substitution rate of  <dig>  × 10- <dig> substitutions per site per year  <cit> , we estimate the mean insertion time for elements is ~ <dig>  million years ago.
one interesting feature of this analysis was that there were very few short tbls , which suggests a lack of recent helitron activity.
the overall impression is that of a large family of helitron elements in rice that have not been particularly active in the very recent past.
we assessed polymorphism of rice helitrons computationally by comparing their locations between the japonica and indica genomes.
to first determine orthology between helitrons, we compared  <dig> bp flanking each side of each helitron between genomes .
assuming no genomic rearrangements between japonica and indica, orthologous helitrons should not only share flanking regions but also be in the same chromosome, on the same strand and between the same bordering flanking regions.
given this definition of orthology and a subsequent list of possible orthologous pairings, we used the maximal non-crossing matching algorithm  <cit>  to find the one-to-one mapping with the largest cardinality.
using this approach, we estimate that  <dig> of the  <dig> japonica  elements are found in the same location within the indica genome.
for the 27% of japonica helitrons that were not found in the same location indica, it was not always clear if the helitron is truly polymorphic between genomes or if data were missing in one of the two genome sequences.
however, some helitrons were polymorphic between japonica and indica with insertion sites at the predicted 3' and 5' boundaries , as might be expected of recently inserted elements.
thus, our 27% estimate contains some true polymorphisms, but this figure is also the upper limit of helitron polymorphism between the japonica and indica genomes.
the true value could be substantially lower.
characterization of captured coding sequences
we compared our sample of maize and japonica rice helitrons to three data sources – plant ests, plant cdss, and plant genomic data – to identify gene capture events.
the maize helitrons had hundreds of hits to est data, but it was not always possible to determine whether this was due to homology to a known protein, due to transcription of portions of the helitron, or due to other unknown factors.
we thus adopted conservative criteria to identify gene capture events: i) matches were only scored with length criteria  at stringent cutoffs  and ii) gene capture was only noted if homology with cds or genomic data included a clearly annotated gene or exon.
even with these conservative criteria, gene capture is clearly rampant within maize, as noted previously  <cit> .
we identified  <dig> genes that had been captured by helitrons .
many of the capture events were shared among our predicted and 'seed' helitrons, suggesting that our search yielded predicted elements with internal similarities to confirmed helitrons.
multiple exons were captured for several of the genes.
as these capture events were duplicated via transposition, some portions of the capture event were lost from template copies, resulting in helitrons that retain different portions of the same gene capture event.
for example, a  <dig> bp piece of the oleosin kd <dig> gene has been captured in helitrons  <dig>   <dig>   <dig>  and  <dig>  but only the last  <dig> bp of this sequence is present in helitrons  <dig> and  <dig>  while helitron  <dig> contains only the first  <dig> bp .
* number of helitrons containing a fragment from that gene.
in stark contrast to maize, we found evidence for only  <dig> unique gene capture events in the  <dig> japonica rice helitrons.
portions of these  <dig> genes are found in 92%  of the predicted elements.
six of the  <dig> genes are distributed widely, in that portions of each of these genes can be detected in ~ <dig> elements .
it also is not uncommon for a helitrons to contain genic fragments that originated from more than one of the eleven genes – i.e., 61%  of the predicted elements house genic fragments that originated from two genes.
for example, portions of both the dextrinase gene and the ribosomal s <dig> gene are found in  <dig> elements.
overall, these data paint a picture of rice helitrons in which gene capture is infrequent but individual capture events are distributed by transposition  throughout the genome.
in this respect, our sample of non-autonomous rice helitrons are more similar to those in arabidopsis than those in maize.
only five gene capture events were identified in  <dig> arabidopsis helitrons, but they too were distributed in many copies throughout the genome  <cit> .
* number of helitrons containing a fragment from that gene.
comparative analysis of 3' palindrome folding properties
maize helitrons contain more vestiges of genes than either rice or arabidopsis helitrons; we postulated that this difference could be mechanistic and perhaps reflected by sequence characteristics among species.
more specifically, 3' palindromes of helitrons are hypothesized to play a role in replication termination  <cit> .
if this is true, we predicted that rice would have stronger 3' palindromes, facilitating less "leaky" termination, commensurately less incorporation of flanking nucleotides during replication, and therefore less gene capture.
we tested this hypothesis by isolating  <dig> bp of the 3' of each of our helitrons in maize and japonica rice and submitting them to unafold analysis to estimate the average free energy of folding .
contrary to our prediction, the  <dig> predicted japonica rice helitrons have dg¯ values significantly higher than that of our collection of maize helitrons, with dg¯ = - <dig>   and dg¯ = - <dig>   for rice and maize, respectively .
the difference remains significant whether one compares polymorphic helitrons between maize and rice  or fixed helitrons between maize and rice .
in contrast there is no significant difference in dg¯ between polymorphic and fixed helitrons within either species .
thus, there are differences in 3' folding characteristics between maize and rice, but the direction of the difference was contrary to our prediction in that the rice elements have weaker 3' palindromes on average.
discussion
non-autonomous helitrons can be difficult to identify both because their internal sequences are not well conserved and because they lack the obvious features of some class i and class ii elements.
we pursued two approaches to identify helitrons in maize, both of which were based on the ~ <dig> bp 3' and 5' elements ends.
an iterative, blast-based method yielded a non-redundant set of  <dig> predicted maize helitrons.
our predicted set of maize helitrons is smaller than that of du et al.
<cit> , for two reasons.
first, we may have searched an earlier version of genbank .
second, du et al.
<cit>  used perl regular expression searches based on  <dig> and  <dig> bp queries for the 5' and 3' ends, respectively, which is shorter than many of our blast hits and may not be sufficient for appropriate identification of helitrons  <cit> .
nonetheless,  <dig> bp and  <dig> bp queries do appear to adequately cover the length of conserved 5' and 3' motifs .
we used our set of maize  and similarly identified rice helitrons to construct profile hmms for each terminal region and each species.
the rice hmms ranked the rice training sequences near the top of the list of all potential genome hits and identified >  <dig> elements in each of the japonica and indica genomes, representing the first genome-wide description of putative helitrons in rice.
five features of this sequence collection suggest they represent bona fide rice helitrons: i) the end sequences feature known hallmarks of helitron 3' and 5' ends ; ii) the 3' and 5' ends are found much more often than expected in random sequences; iii) some elements are polymorphic between japonica and indica ; iv) the elements share internal similarities, even though internal similarity was not a search criterion; and v) the putative rice helitrons contain gene fragments, like helitrons in other plant species.
the effectiveness of the rice hmms may be due to the presence of a large, 508-member subfamily with similar sequence properties that provide a template for hmm convergence.
in contrast, maize hmms failed to achieve an acceptable rate of recall and did not effectively model the 5' and 3' motifs.
one potential explanation for the failure of the maize hmm is the limited maize genomic data available at the time of the analysis.
however we suspect, but cannot prove, that the failure of hmms to converge reflects a broader diversity of helitrons in maize than rice, with perhaps more subfamilies of fewer members.
this conjecture is supported by the fact that single-linkage clustering of our maize elements yielded a largest cluster of only  <dig> elements, with  <dig> of  <dig> elements remaining as singletons, suggesting maize helitrons share few extensive internal sequence characteristics.
nonetheless, our number of predicted helitrons in maize is sufficient to categorize substantial differences among species.
first, at an average length of  <dig> bp, maize helitrons are longer than those of the other two species, with average lengths of  <dig> bp and  <dig> bp for rice and arabidopsis, respectively.
second, both rice and arabidopsis have clear subfamily structure.
this study identified at least one major subfamily of non-autonomous elements in rice, with >  <dig> elements.
although several distinct subfamilies of non-autonomous helitrons have been identified in arabidopsis  <cit> , a subfamily >  <dig> members predominates the genomic landscape  <cit> .
the lack of maize clusters with >  <dig> elements may be in part an effect of small sample, but nonetheless the difference in single-linkage clusters among species is suggestive that patterns of helitron diversity vary substantially among angiosperm species.
helitron polymorphism among individuals
it is also useful to compare polymorphism among individuals across maize, rice and arabidopsis.
the  <dig> 'seed' helitrons in maize were identified by virtue of the fact that they were polymorphic among individuals, so this was not a random set and cannot be considered to provide an unbiased window into the rate of helitron presence/absence polymorphism.
we did find, however, that five of our maize helitrons were polymorphic in a sample of  <dig> inbred lines, out of  <dig> predicted helitrons for which we were able to procure clean and repeatable pcr results.
in rice, we found a maximal polymorphism rate of 27% between japonica and indica, and in a. thaliana, 51% of  <dig> non-autonomous elements were polymorphic in a sample of  <dig> individuals  <cit> .
the percentage of polymorphic elements is not directly comparable among species because the percentage is an increasing function of the number of individuals assayed for polymorphism.
however, one can compare diversity with watterson's θ  <cit> , which corrects for sample size and can be used as an estimate of per element helitron polymorphism among individuals.
the estimate of θ for rice is  <dig> , while that of maize and arabidopsis are similar at  <dig>  and  <dig> , respectively.
as noted above, the value for rice is undoubtedly inflated by incomplete sequence data between japonica and indica rice genomes.
indeed, long tbls in the rice helitron phylogenies suggest that few element insertions have been recent , suggesting that few helitrons should be polymorphic between japonica and indica.
nonetheless, the fact that θ per element is similar between maize and arabidopsis suggests that: i) our helitron predictions yield reasonable answers, in terms of the polymorphism levels of non-autonomous helitrons polymorphism in maize relative to the much better characterized genome of arabidopsis and/or ii) maize helitrons may not be exceptionally polymorphic among individuals relative to helitrons in other angiosperm species.
gene capture
perhaps the most puzzling difference among species is the extent of gene capture.
the propensity for maize helitrons to capture gene fragments is well known  <cit> ; our additional analyses of gene capture events only serve to corroborate these earlier observations .
in contrast, the relative dearth of gene fragments in non-autonomous helitrons has been characterized in a. thaliana and now rice .
although gene capture is a predominant feature of maize helitrons, it may not be a common feature of plant helitrons sensu lato.
why is gene capture common in maize, relative to arabidopsis and rice?
one obvious consideration is sampling biases.
as described above, the complement of non-autonomous helitrons described to date in both arabidopsis and rice are dominated by a single subfamily.
it is possible that gene capture is for some reason particularly rare in these subfamilies, and that helitrons representing gene capture events have escaped identification by the different methods used in this study and the study of arabidopsis  <cit> .
another sampling phenomenon could apply to maize, in that our sample of maize helitrons may be non-representative, thereby over-representing the prevalence of gene capture.
yet, this also seems unlikely because previously described, polymorphic helitrons also contain a number of gene fragments  <cit> .
another possibility for differences in gene capture, along with sampling phenomena, is time.
yet, the effects of time are difficult to predict precisely.
one the one hand, if helitrons are older in maize, then there may have been more time for gene capture events to occur.
on the other hand , it might be reasonable to expect younger elements to contain more captured gene fragments.
the reasoning here is that captured gene fragments, which can be expressed  <cit> , might be particularly deleterious for genome function.
if that is generally the case, non-autonomous helitrons with gene fragments will be preferentially removed from the genome over helitrons that do not harbor expressed gene fragments.
that is, older elements should have fewer gene fragments.
although the predicted relationship between time and gene capture is unclear, several features of our data suggest that time does not drive the observed differences among species.
on the one hand, many  of the maize helitrons are new insertions, given that they are polymorphic among inbred lines.
however, arabidopsis helitrons have similar levels of polymorphism among individuals and thus are also recent.
arabidopsis elements have amplified within  <dig> mya  <cit> , after the divergence of a. thaliana from its sister species a. lyrata  <cit> .
yet, despite potentially similar time dynamics, maize and arabidopsis helitrons differ markedly in the number of gene capture events.
moreover, rice and arabidopsis helitrons have comparable numbers of gene capture events, but helitrons in these two species seem to differ dramatically in terms of evolutionary time, with the rice elements providing little evidence of recent transposition events based on tbls .
a third possible contributor to the difference in gene capture is differences in the fidelity of replication mechanisms.
we proposed that the properties of 3' palindromes could differ between species, thinking that the species with weaker palindromes would have 'leakier' replication that resulted in the poor termination of replication and accompanying incorporation of flanking genes.
we did find a difference in the free energy of folding between maize and rice, but it differed in direction from our prediction; maize helitrons have stronger 3' palindromes than rice.
this observation is not easy to reconcile with our prediction, but does call to question the functional significance of 3' palindromes.
the functional roll of palindromes in the termination of replication is at this point hypothetical  <cit> .
in fact, brunner et al  <cit>  suggest that the 3' end of helitrons plays a role in the initiation  of transposition.
they base this argument on their observation that 3' ends appear to be more conserved than 5' ends; this observation is supported, but only nominally, by our 3' and 5' nucleotide profiles .
if the 3' ends serve to initiate transposition, one might predict that more recent and active elements will have strong palindromes, but we detect no difference in dg¯ between helitrons that are recent  and older .
added to this mystery, helitrons have been found recently without 3' palindromes  <cit> , suggesting that the 3' hairpins may not be necessary for element function at all.
to address bioinformatically the issue of 3' palindrome function, we asked whether the 3' palindromes of maize helitrons had unique properties relative to the complete helitron sequence.
to do this, we examined all  <dig> maize helitrons, scanned for hairpins along their length , estimated dg for each hairpin, and graphed dg in relation to length.
even counting liberally, only a small subset of nine of  <dig> helitrons have 3' hairpins with markedly low dg properties relative to other hairpins in the same helitron sequence .
these nine helitrons are not biased as to whether the element is or is not demonstrably polymorphic among individuals .
another nine helitron sequences had 3' hairpin structures of moderate folding strength relative to palindromes throughout the remainder of the sequence, and  <dig> helitrons had 3' hairpins without an obviously strong 3' hairpin .
we thus find little evidence to suggest that the 3' palindromes are particularly unique within individual helitron sequences, suggesting that 3' palindromes either may not be necessary for helitron function or, at the very least, must act in concert with other signals, such as the terminal ctag.
CONCLUSIONS
why do maize helitrons capture so many genes relative to rice and arabidopsis?
it is hard to make convincing arguments for time, enhanced polymorphism levels among individuals, or properties of 3' hairpins, and thus the answer to this question remains a mystery.
one factor that we cannot address properly here is genomic defense; both rice and arabidopsis have small genomes, and they may more effectively purge long elements  to maintain genome size  <cit> .
another possibility is that maize has more total transposition events.
the number of transposition events is the product of the potentially mobile elements  along with the transposition rate per element  <cit> .
with more events, there is more opportunity for gene capture.
there is, however, currently no data to argue for or against the idea that maize has had more transposition events in its recent evolutionary history than either maize or arabidopsis.
a third possibility is that gene capture leads almost immediately to new helitron subfamilies.
each new subfamily then may represent a new te invasion event  <cit>  that can escape genomic defenses.
hence, once gene capture begins, it can become a run-away process, but then the mystery is how it begins.
unfortunately, there is currently not enough information about helitron copy number in maize, the diversity of helitrons in maize, or the transposition rates of helitrons in any species to make definitive conclusions.
additional analysis of the full maize genome sequence will provide some insights, but additional information about transposition rates and population dynamics will also be critical for understanding the striking differences among helitrons of different angiosperm species.

 traditionally, transposable elements have been classified as either class i or class ii. class i, or retroelements, transpose through an rna intermediate. this group includes both long-terminal repeat transposons and non-long-terminal repeat retrotransposons. class ii elements transpose via dna, include inverted repeats, and often leave a "footprint" after excision. the two classes of transposable elements share at least two features: first, both duplicate host sequences during integration into the host genome, and second, the 3' end of the insertion is either a duplicate of the 5' end or a poly tail. in kapitonov and jurka identified a new group of eukarotyic dna tes in eukaryotes that they named helitrons. helitrons are dna elements that are proposed to move via a rolling circle replication mechanism similar to that of some prokaryotic transposable elements. they are unique in that they do not duplicate host insertion sites and do not contain terminal repeats. they can also be quite large, up to kb or more. however, helitrons do have two conserved sequence features. first, they tend to insert between adenine and thymidine residues. second, they have conserved ends consisting of tc on the 5' end and ctrr on the 3' end, often with a palindromic sequence of 16– bp near the 3' terminus. because they do not replicate by the "cut and paste" method of most class ii elements, helitrons were initially difficult to classify and were considered their own class. they have now been assigned as a separate subclass of class ii tes. non-autonomous helitrons are common in eukaryotic genomes, representing ~2–3% of genomes in species as diverse as arabidopsis thaliana, caenorabditis elegans and bats, but they are arguably most interesting in maize, where they contribute to substantial differences in gene order and content among individuals. for example, the bronze region features extensive sequence non-homologies among maize inbred lines, in large part due to non-autonomous helitron insertions. remarkably, much of this non-homology represents expressed gene fragments that have been captured within non-autonomous elements. gene capture appears to be both widespread and frequent. for example, just four helitrons were found to contain fragments that originated from at least seven genes. moreover, overgo hybridization suggests that as many as genes or gene fragments may be unshared between inbred lines, perhaps due to helitron activity. the presence of expressed gene fragments within helitrons highlights their potential to contribute to functional, as well as structural, diversity within plant genomes. the observations in maize provide a puzzling contrast to observations in arabidopsis. arabidopsis contains > non-autonomous helitrons, many of which were initially catalogued as basho elements. like helitrons in maize, many a. thaliana elements contain gene fragments. unlike maize, however, these fragments appear to represent only a small number of gene capture events. this contrast between maize and arabidopsis raises a number of important questions about the evolution of helitrons and their role in gene capture. first, are the small numbers of maize helitrons characterized thus far typical with respect to the extent of gene capture, or could the apparent differences between species be due to the small number of helitrons isolated from maize thus far? second, if one examines a third experimental system, like rice, do maize or arabidopsis helitrons appear to be more typical with respect to gene capture? third, if there are real differences in gene capture between maize and other plant species, can one glean any clues as to the mechanistic or evolutionary factors that contribute to these differences?  helitron identification and polymorphism in maize we used a blast-based approach to identify helitrons from maize genomic data. our search began with a training set of maize'seed' elements from the literature, from which we isolated both 5' and 3' ends as blast queries. these queries were bp in length, but generally yielded ~ bp matches corresponding to helitron ends. this approach was applied iteratively, accruing additional blast queries, until no more ends were detected. after careful consideration of redundancy in genbank entries, we produced a curated set of predicted maize helitrons, with a mean length of base pairs and a range from bp to bp. we combined our predicted helitrons with the query sequences to produces a total set of maize helitrons. before drawing conclusions from our predicted set of maize helitrons, it is important to examine the properties of the predicted elements. we first examined the 3' and 5' ends for features characteristic of helitrons. to date, maize helitrons have had 5' ends marked by insertion next to a flanking'a' and a beginning sequence of'tct', followed by a bp pattern without a strong consensus. du et al. recently used this bp pattern, including sequence ambiguities, to identify potential 5' ends of helitrons in maize genomic sequence. our collection of putative elements perfectly reflect the search pattern of du et al., but also clearly indicate a lack of strong consensus nucleotides at residues to. similarly, our sample of predicted helitrons reflect the recognized preference for helitrons to terminate in'ctagt', and the 3' ~ bp motif also match the maize consensus. although our search began with a seed set that reflected particular properties, there is no guarantee that our blast strategy would return strong signals in the particular residues that have previously been defined as typical of helitrons. moreover, these patterns are qualitatively indistinguishable when the'seed' helitrons are removed from consideration. in addition to 5' and 3' motifs, our results clearly indicate the lack of consensus nucleotides in flanking genomic sequences, except for the strong bias toward inserting downstream from an'a'. because maize helitrons were first identified by comparisons of sequence data between maize inbred lines, we next assessed polymorphism among maize individuals. we attempted to survey our predicted helitrons with a pcr assay of eight maize inbred lines. we were unable to amplify single bands consistently for eight of the predicted helitrons, and so could not unambiguously assess polymorphism for these presumptive helitrons. another eight were fixed in the panel of eight maize inbred lines, and polymorphism could not be verified in a broader germplasm set, either due to a lack of polymorphism or poor amplification properties. however, five helitrons were polymorphic in the sample of inbred lines. thus, of our predicted helitrons were confirmed to be polymorphic. we next used the 5' and 3' ends from our panel of putative maize helitrons to build profile hmm models and used the models to scan all maize genomic sequences in genbank. we found that the recall rate, which is a measure of sensitivity, was very low, indicating that the hmm could not effectively model the 5' and 3' maize motifs. the failure of the hmm approach may indicate that maize harbors a broad diversity of helitron subfamilies with widely variable 3' and 5' ends. helitron identification and polymorphism in rice while conducting our blast-based search for maize helitrons in genbank, we also discovered putative helitrons in genomic rice sequences. mirroring our approach for maize, we used the 5' and 3' ends from this panel of putative rice helitrons to build profile hmms and used the models to scan two separate sources: genomic sequences of indica and japonica rice. in contrast to the hmm application to maize, the rice hmms achieved a much higher recall rate, suggesting that rice helitrons may share extensive sequence similarity. the hmm models identified putative helitron elements in the japonica rice genome, and elements in the indica rice genome. to determine whether we would expect to identify elements based on chance alone, we constructed randomized rice genomes with the same length and compositional properties of japonica rice and reapplied the hmm. in five randomized genomes, the hmm failed to identify a single helitron, thus illustrating that our findings are not expected due to random hits to a genomic sequence of similar length and composition. because the helitron components from the rice genomes were similar and because the japonica genome is better annotated, we focused on the set of japonica elements for description. the mean length of predicted japonica helitrons was bp, with 97% of these under kb in length and at least two of the longest containing internal retrotransposons. none of the putative helitrons appear to be autonomous, based both on length and on blastn comparisons to putative helicase genes. we examined the 5' and 3' ends of predicted rice helitrons, just as we examined them in maize. the rice helitrons generally yield stronger consensus motifs, with nucleotide biases extending for ~ bp on the 5' end and bp on the 3' end. nonetheless, there are shared features of the motifs between maize and rice, particularly the 5''tct' and the 3''ctagt'. we also examined the regions flanking rice helitrons. like maize, the helitrons exhibited a preference for a 5' flanking'a' nucleotide. the 3' flanking sequences were markedly a+t rich, which is also a preference in maize but less pronounced. the strong motif pattern in rice terminal regions suggested that we had uncovered a well-conserved group of helitrons. to pursue this suspicion, we separated the putative helitrons into single-linkage clusters, yielding one particularly large cluster of elements for which we constructed a neighbor-joining tree. with this tree, we were able to infer approximate times of element insertion. using the terminal branch lengths in the phylogeny as a proxy for time, and assuming a molecular clock with a nucleotide substitution rate of × 10- substitutions per site per year, we estimate the mean insertion time for elements is ~ million years ago. one interesting feature of this analysis was that there were very few short tbls, which suggests a lack of recent helitron activity. the overall impression is that of a large family of helitron elements in rice that have not been particularly active in the very recent past. we assessed polymorphism of rice helitrons computationally by comparing their locations between the japonica and indica genomes. to first determine orthology between helitrons, we compared bp flanking each side of each helitron between genomes. assuming no genomic rearrangements between japonica and indica, orthologous helitrons should not only share flanking regions but also be in the same chromosome, on the same strand and between the same bordering flanking regions. given this definition of orthology and a subsequent list of possible orthologous pairings, we used the maximal non-crossing matching algorithm to find the one-to-one mapping with the largest cardinality. using this approach, we estimate that of the japonica elements are found in the same location within the indica genome. for the 27% of japonica helitrons that were not found in the same location indica, it was not always clear if the helitron is truly polymorphic between genomes or if data were missing in one of the two genome sequences. however, some helitrons were polymorphic between japonica and indica with insertion sites at the predicted 3' and 5' boundaries, as might be expected of recently inserted elements. thus, our 27% estimate contains some true polymorphisms, but this figure is also the upper limit of helitron polymorphism between the japonica and indica genomes. the true value could be substantially lower. characterization of captured coding sequences we compared our sample of maize and japonica rice helitrons to three data sources – plant ests, plant cdss, and plant genomic data – to identify gene capture events. the maize helitrons had hundreds of hits to est data, but it was not always possible to determine whether this was due to homology to a known protein, due to transcription of portions of the helitron, or due to other unknown factors. we thus adopted conservative criteria to identify gene capture events: i) matches were only scored with length criteria at stringent cutoffs and ii) gene capture was only noted if homology with cds or genomic data included a clearly annotated gene or exon. even with these conservative criteria, gene capture is clearly rampant within maize, as noted previously. we identified genes that had been captured by helitrons. many of the capture events were shared among our predicted and'seed' helitrons, suggesting that our search yielded predicted elements with internal similarities to confirmed helitrons. multiple exons were captured for several of the genes. as these capture events were duplicated via transposition, some portions of the capture event were lost from template copies, resulting in helitrons that retain different portions of the same gene capture event. for example, a bp piece of the oleosin kd gene has been captured in helitrons and but only the last bp of this sequence is present in helitrons and while helitron contains only the first bp. * number of helitrons containing a fragment from that gene. in stark contrast to maize, we found evidence for only unique gene capture events in the japonica rice helitrons. portions of these genes are found in 92% of the predicted elements. six of the genes are distributed widely, in that portions of each of these genes can be detected in ~ elements. it also is not uncommon for a helitrons to contain genic fragments that originated from more than one of the eleven genes – i.e., 61% of the predicted elements house genic fragments that originated from two genes. for example, portions of both the dextrinase gene and the ribosomal s gene are found in elements. overall, these data paint a picture of rice helitrons in which gene capture is infrequent but individual capture events are distributed by transposition throughout the genome. in this respect, our sample of non-autonomous rice helitrons are more similar to those in arabidopsis than those in maize. only five gene capture events were identified in arabidopsis helitrons, but they too were distributed in many copies throughout the genome. * number of helitrons containing a fragment from that gene. comparative analysis of 3' palindrome folding properties maize helitrons contain more vestiges of genes than either rice or arabidopsis helitrons; we postulated that this difference could be mechanistic and perhaps reflected by sequence characteristics among species. more specifically, 3' palindromes of helitrons are hypothesized to play a role in replication termination. if this is true, we predicted that rice would have stronger 3' palindromes, facilitating less "leaky" termination, commensurately less incorporation of flanking nucleotides during replication, and therefore less gene capture. we tested this hypothesis by isolating bp of the 3' of each of our helitrons in maize and japonica rice and submitting them to unafold analysis to estimate the average free energy of folding. contrary to our prediction, the predicted japonica rice helitrons have dg¯ values significantly higher than that of our collection of maize helitrons, with dg¯ = - and dg¯ = - for rice and maize, respectively. the difference remains significant whether one compares polymorphic helitrons between maize and rice or fixed helitrons between maize and rice. in contrast there is no significant difference in dg¯ between polymorphic and fixed helitrons within either species. thus, there are differences in 3' folding characteristics between maize and rice, but the direction of the difference was contrary to our prediction in that the rice elements have weaker 3' palindromes on average. discussion non-autonomous helitrons can be difficult to identify both because their internal sequences are not well conserved and because they lack the obvious features of some class i and class ii elements. we pursued two approaches to identify helitrons in maize, both of which were based on the ~ bp 3' and 5' elements ends. an iterative, blast-based method yielded a non-redundant set of predicted maize helitrons. our predicted set of maize helitrons is smaller than that of du et al., for two reasons. first, we may have searched an earlier version of genbank. second, du et al. used perl regular expression searches based on and bp queries for the 5' and 3' ends, respectively, which is shorter than many of our blast hits and may not be sufficient for appropriate identification of helitrons. nonetheless, bp and bp queries do appear to adequately cover the length of conserved 5' and 3' motifs. we used our set of maize and similarly identified rice helitrons to construct profile hmms for each terminal region and each species. the rice hmms ranked the rice training sequences near the top of the list of all potential genome hits and identified > elements in each of the japonica and indica genomes, representing the first genome-wide description of putative helitrons in rice. five features of this sequence collection suggest they represent bona fide rice helitrons: i) the end sequences feature known hallmarks of helitron 3' and 5' ends; ii) the 3' and 5' ends are found much more often than expected in random sequences; iii) some elements are polymorphic between japonica and indica; iv) the elements share internal similarities, even though internal similarity was not a search criterion; and v) the putative rice helitrons contain gene fragments, like helitrons in other plant species. the effectiveness of the rice hmms may be due to the presence of a large, 508-member subfamily with similar sequence properties that provide a template for hmm convergence. in contrast, maize hmms failed to achieve an acceptable rate of recall and did not effectively model the 5' and 3' motifs. one potential explanation for the failure of the maize hmm is the limited maize genomic data available at the time of the analysis. however we suspect, but cannot prove, that the failure of hmms to converge reflects a broader diversity of helitrons in maize than rice, with perhaps more subfamilies of fewer members. this conjecture is supported by the fact that single-linkage clustering of our maize elements yielded a largest cluster of only elements, with of elements remaining as singletons, suggesting maize helitrons share few extensive internal sequence characteristics. nonetheless, our number of predicted helitrons in maize is sufficient to categorize substantial differences among species. first, at an average length of bp, maize helitrons are longer than those of the other two species, with average lengths of bp and bp for rice and arabidopsis, respectively. second, both rice and arabidopsis have clear subfamily structure. this study identified at least one major subfamily of non-autonomous elements in rice, with > elements. although several distinct subfamilies of non-autonomous helitrons have been identified in arabidopsis, a subfamily > members predominates the genomic landscape. the lack of maize clusters with > elements may be in part an effect of small sample, but nonetheless the difference in single-linkage clusters among species is suggestive that patterns of helitron diversity vary substantially among angiosperm species. helitron polymorphism among individuals it is also useful to compare polymorphism among individuals across maize, rice and arabidopsis. the'seed' helitrons in maize were identified by virtue of the fact that they were polymorphic among individuals, so this was not a random set and cannot be considered to provide an unbiased window into the rate of helitron presence/absence polymorphism. we did find, however, that five of our maize helitrons were polymorphic in a sample of inbred lines, out of predicted helitrons for which we were able to procure clean and repeatable pcr results. in rice, we found a maximal polymorphism rate of 27% between japonica and indica, and in a. thaliana, 51% of non-autonomous elements were polymorphic in a sample of individuals. the percentage of polymorphic elements is not directly comparable among species because the percentage is an increasing function of the number of individuals assayed for polymorphism. however, one can compare diversity with watterson's θ, which corrects for sample size and can be used as an estimate of per element helitron polymorphism among individuals. the estimate of θ for rice is, while that of maize and arabidopsis are similar at and, respectively. as noted above, the value for rice is undoubtedly inflated by incomplete sequence data between japonica and indica rice genomes. indeed, long tbls in the rice helitron phylogenies suggest that few element insertions have been recent, suggesting that few helitrons should be polymorphic between japonica and indica. nonetheless, the fact that θ per element is similar between maize and arabidopsis suggests that: i) our helitron predictions yield reasonable answers, in terms of the polymorphism levels of non-autonomous helitrons polymorphism in maize relative to the much better characterized genome of arabidopsis and/or ii) maize helitrons may not be exceptionally polymorphic among individuals relative to helitrons in other angiosperm species. gene capture perhaps the most puzzling difference among species is the extent of gene capture. the propensity for maize helitrons to capture gene fragments is well known; our additional analyses of gene capture events only serve to corroborate these earlier observations. in contrast, the relative dearth of gene fragments in non-autonomous helitrons has been characterized in a. thaliana and now rice. although gene capture is a predominant feature of maize helitrons, it may not be a common feature of plant helitrons sensu lato. why is gene capture common in maize, relative to arabidopsis and rice? one obvious consideration is sampling biases. as described above, the complement of non-autonomous helitrons described to date in both arabidopsis and rice are dominated by a single subfamily. it is possible that gene capture is for some reason particularly rare in these subfamilies, and that helitrons representing gene capture events have escaped identification by the different methods used in this study and the study of arabidopsis. another sampling phenomenon could apply to maize, in that our sample of maize helitrons may be non-representative, thereby over-representing the prevalence of gene capture. yet, this also seems unlikely because previously described, polymorphic helitrons also contain a number of gene fragments. another possibility for differences in gene capture, along with sampling phenomena, is time. yet, the effects of time are difficult to predict precisely. one the one hand, if helitrons are older in maize, then there may have been more time for gene capture events to occur. on the other hand, it might be reasonable to expect younger elements to contain more captured gene fragments. the reasoning here is that captured gene fragments, which can be expressed, might be particularly deleterious for genome function. if that is generally the case, non-autonomous helitrons with gene fragments will be preferentially removed from the genome over helitrons that do not harbor expressed gene fragments. that is, older elements should have fewer gene fragments. although the predicted relationship between time and gene capture is unclear, several features of our data suggest that time does not drive the observed differences among species. on the one hand, many of the maize helitrons are new insertions, given that they are polymorphic among inbred lines. however, arabidopsis helitrons have similar levels of polymorphism among individuals and thus are also recent. arabidopsis elements have amplified within mya, after the divergence of a. thaliana from its sister species a. lyrata. yet, despite potentially similar time dynamics, maize and arabidopsis helitrons differ markedly in the number of gene capture events. moreover, rice and arabidopsis helitrons have comparable numbers of gene capture events, but helitrons in these two species seem to differ dramatically in terms of evolutionary time, with the rice elements providing little evidence of recent transposition events based on tbls. a third possible contributor to the difference in gene capture is differences in the fidelity of replication mechanisms. we proposed that the properties of 3' palindromes could differ between species, thinking that the species with weaker palindromes would have'leakier' replication that resulted in the poor termination of replication and accompanying incorporation of flanking genes. we did find a difference in the free energy of folding between maize and rice, but it differed in direction from our prediction; maize helitrons have stronger 3' palindromes than rice. this observation is not easy to reconcile with our prediction, but does call to question the functional significance of 3' palindromes. the functional roll of palindromes in the termination of replication is at this point hypothetical. in fact, brunner et al suggest that the 3' end of helitrons plays a role in the initiation of transposition. they base this argument on their observation that 3' ends appear to be more conserved than 5' ends; this observation is supported, but only nominally, by our 3' and 5' nucleotide profiles. if the 3' ends serve to initiate transposition, one might predict that more recent and active elements will have strong palindromes, but we detect no difference in dg¯ between helitrons that are recent and older. added to this mystery, helitrons have been found recently without 3' palindromes, suggesting that the 3' hairpins may not be necessary for element function at all. to address bioinformatically the issue of 3' palindrome function, we asked whether the 3' palindromes of maize helitrons had unique properties relative to the complete helitron sequence. to do this, we examined all maize helitrons, scanned for hairpins along their length, estimated dg for each hairpin, and graphed dg in relation to length. even counting liberally, only a small subset of nine of helitrons have 3' hairpins with markedly low dg properties relative to other hairpins in the same helitron sequence. these nine helitrons are not biased as to whether the element is or is not demonstrably polymorphic among individuals. another nine helitron sequences had 3' hairpin structures of moderate folding strength relative to palindromes throughout the remainder of the sequence, and helitrons had 3' hairpins without an obviously strong 3' hairpin. we thus find little evidence to suggest that the 3' palindromes are particularly unique within individual helitron sequences, suggesting that 3' palindromes either may not be necessary for helitron function or, at the very least, must act in concert with other signals, such as the terminal ctag.  why do maize helitrons capture so many genes relative to rice and arabidopsis? it is hard to make convincing arguments for time, enhanced polymorphism levels among individuals, or properties of 3' hairpins, and thus the answer to this question remains a mystery. one factor that we cannot address properly here is genomic defense; both rice and arabidopsis have small genomes, and they may more effectively purge long elements to maintain genome size. another possibility is that maize has more total transposition events. the number of transposition events is the product of the potentially mobile elements along with the transposition rate per element. with more events, there is more opportunity for gene capture. there is, however, currently no data to argue for or against the idea that maize has had more transposition events in its recent evolutionary history than either maize or arabidopsis. a third possibility is that gene capture leads almost immediately to new helitron subfamilies. each new subfamily then may represent a new te invasion event that can escape genomic defenses. hence, once gene capture begins, it can become a run-away process, but then the mystery is how it begins. unfortunately, there is currently not enough information about helitron copy number in maize, the diversity of helitrons in maize, or the transposition rates of helitrons in any species to make definitive conclusions. additional analysis of the full maize genome sequence will provide some insights, but additional information about transposition rates and population dynamics will also be critical for understanding the striking differences among helitrons of different angiosperm species. 
helitrons are dna transposable elements that are proposed to replicate via a rolling circle mechanism. non-autonomous helitron elements have captured gene fragments from many genes in maize but only a handful of genes in arabidopsis. we performed computational searches to identify helitrons in maize and rice genomic sequence data. we identified over helitrons in the japonica rice genome. the free energy of folding for maize helitrons were significantly lower than those in rice, but the direction of the difference differed from our prediction. our total sample of maize helitrons contained fragments of captured genes. maize helitrons are clearly unique relative to those of rice and arabidopsis in the prevalence of gene capture, but the reasons for this difference remain elusive. maize helitrons do not seem to be more polymorphic among individuals than those of arabidopsis; they do not appear to be substantially older or younger than the helitrons in either species; and our analyses provided little evidence that the 3' hairpin plays a role. more than 70% of these were found in a collinear location in the indica rice genome, and clustered as a single large subfamily.
 helitrons are dna transposable elements that are proposed to replicate via a rolling circle mechanism. non-autonomous helitron elements have captured gene fragments from many genes in maize but only a handful of genes in arabidopsis. this observation suggests very different histories for helitrons in these two species, but it is unclear which species contains helitrons that are more typical of plants.  we performed computational searches to identify helitrons in maize and rice genomic sequence data. using previously identified helitrons as a seed set, we identified helitrons in maize, five of which were polymorphic among a sample of inbred lines. our total sample of maize helitrons contained fragments of captured genes. twenty-one of of these helitrons did not cluster with other elements into closely related groups, suggesting substantial diversity in the maize element complement. we identified over helitrons in the japonica rice genome. more than 70% of these were found in a collinear location in the indica rice genome, and clustered as a single large subfamily. the japonica rice elements contained fragments of only genes, a number similar to that in arabidopsis. given differences in gene capture between maize and rice, we examined sequence properties that could contribute to differences in capture rates, focusing on 3' palindromes that are hypothesized to play a role in transposition termination. the free energy of folding for maize helitrons were significantly lower than those in rice, but the direction of the difference differed from our prediction.  maize helitrons are clearly unique relative to those of rice and arabidopsis in the prevalence of gene capture, but the reasons for this difference remain elusive. maize helitrons do not seem to be more polymorphic among individuals than those of arabidopsis; they do not appear to be substantially older or younger than the helitrons in either species; and our analyses provided little evidence that the 3' hairpin plays a role. 