  since the discovery of transposable elements ( tes ) in corn , 
 dna sequencing has revealed that genomes of eukaryotic organisms are largely comprised 
 of evolutionarily significant tes responsible for creation of considerable genetic 
 diversity .
the movement of transposable 
 elements is either autonomous or dependent on other elements .  
classified according to mode of transposition , 
 class i tes , or retrotransposons , are retroviral - type elements which may or may 
 not have long terminal ( direct ) repeats ( ltrs )
. movement of class i elements necessarily involves 
 an rna inter - mediate in what can appropriately be termed  replicative
retrotransposons are transcribed into rna , 
 and then the reverse transcriptase and integrase make and insert a dna copy at a 
 secondary genomic location
dna transposons ,  but it is important to note that class i retrotransposons 
 are also comprised of dna except during transposition .
transposases permit class 
 ii transposable elements to move by a  cut and paste 
 footprints ,  
 telltale evidence for a previous dna transposon insertion , result from imprecise 
 excision .
class ii transposons characteristically 
 have relatively short inverted repeat sequences near their termini and an excision 
 site at each end recognized by the transposase .
one of the first plant transposons 
 mcclintock described , en1 , or the 
 maize suppressor - mutator ( spm ) , is 
 the original example of a  cacta  class , or superfamily , of transposons .
cacta transposons were thought until recently 
 to be found only in plants , but a similar element was discovered   in the 
 genome of schistosoma mansoni , the causative agent of schistosomiasis .
evidence that retrotransposons account for 
 much of the sugar beet ( beta vulgaris l. ) genome was first obtained by schmidt et al . , 
 who described ( 1 ) repetitive dna sequences in beta vulgaris similar to long interspersed nuclear elements 
 ( lines ) , a type of retrotransposon without ltrs , and ( 2 ) other repetitive dna 
 sequences that resembled ltr retrotransposons of the ty1-copia class .
vulmar1 , 
 a mariner - class transposon in beta 
 vulgaris , , 3 909 bp in length , has 32 bp terminal inverted repeats and 
 carries a single orf that encodes a transposase with a characteristic dde signature 
 motif in a single exon .
our interest in repetitive dna developed 
 from our recent discovery of a number of ltrs and retrotransposon genes as well 
 as a transposase gene in the region between two clusters of core plant genes on 
 a 130 kb sugar beet bac [ 7 , 8 ] .
one gene 
 cluster has an npr1-class disease 
 resistance - potentiating gene adjacent to another core plant gene whose 
 predicted product has high similarity to a heat shock factor protein .
the other cluster consists of a signal 
 peptide calmodulin - binding protein kinase gene located near a ck1-class protein 
 kinase gene . in this communication ,
we report the discovery 
 in beta vulgaris of coe1 , a class ii dna transposase gene within 
 putative ltrs and other features that are characteristic of class i ltr - retrotransposons . 

also , a genome scan of arabidopsis 
 thaliana found a similar arrangement of transposon and retro - element genes 
 on chromosome 4 .
the identification of a sugar 
 beet genome - derived bacterial artificial chromosome ( bac ) carrying the npr1 disease resistance control gene has 
 been previously described   as well as the basic methods used for dna sequence 
 analysis . in this study ,
analysis of the npr1 bac was performed using ltr_struc ( http://www.genetics.uga.edu/retrolab/data/ltr_struc.html ) , 
 repfind ( http://zlab.bu.edu/repfind/form.html ) analysis identified 
 identical direct repeats .
etandem ( http://bioweb.pasteur.fr/seqanal/interfaces/etandem.html ) 
 and einverted ( http://edukon.biologie.uni-konstanz.de/cgi-bin/emboss/einverted ) 
 were used to identify tandem and inverted repeats .
emboss   ( http://emboss.sourceforge.net/ ) 
 was also utilized to identify tandem repeats and inverted repeats .  
repeats were also found using ncbi 's blastprogram ( http://www.ncbi.nlm.nih.gov/blast ) by 
 blast of a contig against itself using blastn . 

a sugar beet expressed sequence tag ( est ) database ( http://genomics.msu.edu/sugarbeet/blast.html ) 
 was employed for nucleotide and protein blasts in order to identify possible functional 
 gene expression .
phylogenetic tree 
 analysis was performed using mega 4 software ( http://www.megasoftware.net/ ) . a new multicopy direct tandem repeat ( mdtr ) within an intronic region within the coe1 transposase gene
was identified by blast of the intron against the entire 130 kb 
 bac and plotting a diagram of repeat versus dna base positions with respect to the 
 bac . in order to find the starting points of the repeats , a window which was of a constant length less than one 
 repeat was used in a sliding window technique .
the first base used in this 
 window was the putative starting point of the first repeat in the mdtr , as 
 determined by the blast output .
this window was blasted against the whole 
 intron . if blast found any repeated dna using this template , the window was actually still within the repeated segment
; 
 therefore , the starting base of the first repeat was deduced to be further 
 towards the 5th end .
the window was then 
 moved a few bases in the 5th direction and the amended sequence was subjected 
 to another blast .
genbank accession ef101866 provides annotation 
 of the 130 kb npr1-carrying bac 
 derived from the sugar beet genome .
conserved microsynteny of four core plant 
 genes was observed with other eudicots ( kuykendall et al . ,
submitted ) . to scan the arabidopsis thaliana genome for a coe1-like element(s ) , each chromosome was individually subjected to 
 ltr_struc analysis , and then each putative ltr - retrotransposon element was examined 
 for both a dna transposase gene and a retrotransposon - like integrase or reverse 
 transcriptase gene within its ltrs .
blast and ltr_struc analyses performed on 
 an annotated 130 kb npr1 gene - carrying sugar beet bac ( genbank accession ef 101866 ) revealed the 
 presence of coe1 , which appears to be 
 a new and unique composite of class i and class ii transposable elements .
us h20 was the source of genomic dna for a sugar beet bacterial artificial chromosome 
 ( bac ) library 8 from which a bac clone carrying the npr1 disease resistance control gene was 
 recently identified .
initially detected by ltr_struc analysis as a ltr - retrotransposon , coe1 , defined as 14.5 kb by two 
 putative 169 bp ltrs , has both an rvt2-domain reverse transcriptase pseudogene and another 
 retroviral - like hypothetical gene .
however , a dna transposase gene was found 
 within its central region ( figure 1 ) . in 
 addition to coe1 , ltr_struc analysis performed on the 130-kb npr1 bac 
 identified at least two other ltr - retrotransposons , briefly : ( 1 ) a copia or ty1-like retroelement , bvrtr1 , which has a reverse transcriptase with 
 active site yvddiif ; and ( 2 ) bvrtr2 , a gypsy or ty3-like retroelement 
 with active site fiddili in its conserved rvt1 domain ( unpublished ) .
precedence in the literature exists for similar yet 
 considerably smaller repetitive dna sequences from sugar beet largely 
 uncharacterized except for genomic distribution .
the question then arises of whether the 
 transposase gene of coe1 represents a 
 class ii transposon inserted into a class i ltr - retrotransposon .
this is probably 
 how coe1 originated . in any case , coe1 has salient features of a class ii 
 dna transposon within a class i retrotransposon ( figure 1 ) as described below .
the transposasegene of coe1 has a predicted protein product that is evidently a cacta 
 superfamily dna transposase as deduced from the results of blast amino acid 
 sequence alignments of the predicted protein product with en / spm - type dna transposons ( figure 2 ) .
evidence for   probable expression of coe1 's transposase 
 gene , or at least a similar gene , were 
 two sugar beet ( expressed sequence tags)(ests )   whose nucleotide sequence aligned 
 by blast with coe1 's dna transposase 
 gene : cf542726 ( e = 0.0 ) and bq595658 ( 2e  96 ) . the coe1 dna transposase gene is flanked by inverted repeats and a cacta 
 sequence motif ( figure 1 ) . in addition to coe1 , the prototypical cacta superfamily transposon
en / spm of corn , tam1 of snapdragon   and seven other tnp2-domain transposons , from various plant species , were compared 
 using megalign .  
figure 2 shows the amino 
 acid sequence alignments obtained with one of two conserved domains .  
cluster 
 analysis of these data ( figure 3 ) , a neighbor joining analysis tree , indicates 
 that the coe1 transposase falls into a 
 group we designate as i subgroup a , with other plant tnp2-domain transposases 
 from arabidopsis thaliana ( bab09502 ) , cleome spinosa ( abd969441 ) , and brassica rapa ( baa85462.1 ) .
another subgroup 
 of group i , b has en / spm of zea mays ( aaa66266 ) and oryza sativa japonica np_001062816 . in 
 the amino acid sequence alignments of this particular conserved region ( figure 2 ) , 
 two other dissimilar groups ( ii and iii ) had the remaining four dna transposases 
 ( figure 3 ) .
the en / spm - like superfamily 
 of plant transposons , exemplified by barbara mcclintock 's suppressor / mutator 
 transposons of corn , has been named cacta for the sequence motif recognized for 
 excision .
conservation is well established over the taxonomic divide between 
 eudicots , and monocots . 

coe1 has a centrally located transposase gene , flanked downstream by a pair of imperfect 51 bp i14 inverted repeat sequences ( 94% match ) separated by only 10 bp , and 
 upstream by another repeated sequence , i24 , 
 that   aligns with i14 with about 75% identity over 41 bp .  
these 
 distal i24/i14 inverted repeats are each 
 flanked by a cacta sequence motif . 

coe1 has 
 a total of three orfs : a retroviral - like hypothetical gene orf1 , the tnp2-domain transposase gene and an 
 apparent rvt2-domain reverse 
 transcriptase pseudogene , orf2 .
coe1 has putative long terminal repeats 
 characteristic of ltr retrotransposons ( figure 1 ) .
the 3.6% sequence divergence 
 in the ltrs is consistent with possibility that the retroelement - like features 
 of coe1 are no longer active .
the 5th end of the coe1 positive strand has a pair of i13 inverted repeats 173 bp apart with 76% match over 190 bp and an 
 internal cactataa sequence motif .
i15 inverted repeats are found downstream 
 of coe1 and these inverted repeats , near 
 another cactataa sequence motif , are 52 bp apart with about 74% match over 51 bp .
orf1 , the first retroelement - like gene of coe1 , encodes , in a single exon , a 
 hypothetical protein for which no significant blast alignment is currently found . 

the predicted protein product of orf1 had initially produced a significant 
 blast with a 
orf2 produced only a relatively weak 
 nucleotide blast alignment ( 2e  25 ) to est bi643401 .
orf2 is apparently a 
 pseudogene since a stop signal occurs in the sequence prior to that part of the 
 sequence that would otherwise encode the active site of an rvt2 domain reverse transcriptase .  
although a sequencing error is possible , it is 
 unlikely ; therefore , it is reasonable to deduce from the sequence data that orf2 
 of coe1 is a reverse transcriptase 
 pseudogene . disregarding the stop signal , the predicted protein product of the rvt2-like gene of coe1 aligned well with other rvt2-domain 
 gene products ( figure 4 ) .
the coe1 
 rvt2 domain reverse transcriptase has a ty1-copia - like yvddiil active site which 
 is most highly conserved in comparison with that of the medicago truncatula accession ( figure 4 ) . among the protein alignments 

performed , the hypothetical sugar beet rvt2-domain 
 containing gene product also showed higher similarity with retrotransposon - type 
 reverse transcriptase proteins encoded by genes in two subspecies ( indica and japonica ) of oryza sativa than with most others from 
 a wide taxonomic range ( figure 4 ) .
we recently performed a genome - wide scan or 
 survey of the arabidopsis thaliana genome looking specifically for 
 a composite dna transposon within ltr retrotransposon features similar to coe1 , and a similar single tnp2-domain transposase gene flanked by putative 
 ltrs and other retrotransposon - like features was identified , as described below . an arabidopsis thaliana dna transposase gene within putative ltrs and between ltr - retrotransposon genes
the apparent ltr - retrotransposon is 9 078 bp and has 506 bp 
 and 471 bp ltr regions with about 90% identity .
this element was found on bac t26n6 from chromosome iv at 19.3 cm 
 ( accession af07243 ) .
the first orf ( at4g04426 ) appears to be a highly degraded pseudogene of a reverse transcriptase , the central 
 orf ( at4g04430 ) is a cacta - class transposase gene , and the third orf ( at4g04440 ) has a predicted protein product with an rvt2 reverse 
 transcriptase domain similar to ty1-copia - like retrotransposons .
alignment of a conserved region of the predicted 
 product of the reverse transcriptase and a neighbor joining tree from cluster analysis ( not shown ) revealed a number of similar 
 blast hits but none of these was very nearly identical to the predicted product of the arabidopsis element .
the 
 finding of a coe1-like element in arabidopsis supports the concept that coe1 in 
 beta vulgaris is not a unique instance of a class ii transposon within a class i retrotransposon . regarding the subject of coe1 in beta vulgaris , it is noteworthy that within the transposase gene most 
 of the dna sequence of an intron , when blast against itself yielded multipl , 
 relatively small regions of imperfect dna sequence identity .
these results , when graphed , showed 
 a distinct pattern ( figure 6 ) that suggested a whole series of multicopy direct 
 tandem repeats ( mdtrs ) .
repeats of the apparent mdtr found by blast 
 were of different lengths , but the first repeat in each set began at the same 
 position , and the second repeat in each set ended at the same position .
each 
 successive set of repeats was shorter by about the same number of bases .
each repeated dna sequence , aligned 
 in megalign , gave the following consensus sequence : aggaacatgaaacccaaaaaagggct cgaaatggcttagtttctatcattttcattggctaagtgtattaaacttgcttagaatcata caaccatgtagtagaaagtttaaatgagtattttaggacttgcatgagccattagaactt gaaataggcatagaggtaggatta .
an intron within coe1 's transposase gene was thus determined to have 15 complete 173 
 bp mdtr and a partial repeat at the 3 end .
the sequence of this mdtr - carrying intron 
 did not yield any megablast hits against the ncbi database .
one or more individual 
 repeat(s ) could have transposed into the intron followed by duplication(s ) , or possibly 
 the entire series of multicopy tandem direct repeats transposed into the intron 
 altogether .
the latter possibility may 
 be supported by the observation that the last repeat is partial , presumably a 
 deletion .
how did coe1 , or an arrangement of transposable genes that resembles coe1 , originate ?
a dna transposon could have moved into the middle 
 of an ltr - retrotransposon .
in other words , coe1 's 
 central transposase gene , flanked by inverted repeats and cacta sequence 
 motifs , could have transposed into an ltr - retrotransposon .
the cactataa sequence motif , located 16.3 kb apart , outside the putative ltrs near large inverted repeats , could perhaps be 
 extreme boundaries of coe1 instead of 
 the two putative ltrs ( figure 1 ) separated by 14.5 kb .
it is possible that the 
 intact larger and more complex composite transposon could move 
 about using dna transposase .
the ltrs are flanked by pairs of inverted repeat sequences 
 that may be nonautonomous , miniature inverted [ repeat ] sequence transposable 
 elements ( mites ) ( figure 1 ) .
mites , sometimes called  class iii transposons ,  are 
 dependent on dna transposase . the 8-mer sequence motif cactataa flanks the coe1 ltrs near or within relatively 
 large inverted repeats that are perhaps mites .
we hypothesize that originally a class i 
 ltr - retrotransposon inserted between pairs of mites .
then , a class ii dna transposon moved into the 
 middle of the ltr - retrotransposon , and voila , a composite of class ii and class 
 i elements resembling coe1 , at least conceptually . 
 to summarize , coe1 has a tnp2-domain transposase gene flanked by 
 putative ltrs and between two retrotransposon - like genes , all within cactataas 
 near or within pairs of inverted repeats ( figure 1 )
. one may ask ,  what possible selective 
 advantage would a cacta dna transposon within an ltr retrotransposon have over 
 a simple class i or class ii element alone ?  a combination of class i and class 
 ii features may offer little if any selective advantage , and thus such a composite might be unique . 

the finding of a similar gene arrangement in arabidopsis provides a second example of a composite class ii 
 transposon within a class i retrotransposon .
a change in the expression of a gene when placed under the control of another can 
 confer a selective advantage on the host plant .
increased fitness might also be 
 characteristic of the host of a versatile element which can hypothetically   transpose in either of two known mechanisms . 

such versatility could facilitate more rapid genetic change due both to transposition 
 and to subsequent blockage of gene conversion .
in conclusion , based on results of in silico analyses , coe1 , 
 found in the sugar beet genome , can be viewed as an incipient or emerging cacta 
 super - family dna transposon amalgamated within an ltr - retrotransposon .
a similar arrangement of a central tnp2-domain transposase gene within ltrs and between retrotransposon 
 genes was found in chromosome 4 of arabidopsis 
 thaliana by a genome scan .  
more dna sequencing of the sugar beet genome , 
 either of larger stretches or of the complete genome , is likely to be needed in 
 order to distinguish whether coe1 represents an evolutionarily significant gene arrangement or a mere coincidental 
 merging of transposable genes . in either case , as far we know , the two 
 examples shown here of a class ii dna 
 transposon within a class i retrotransposn are novel .
 we describe discovery in beta vulgaris l. of coe1 , a dna transposase gene within putative long terminal repeats ( ltrs ) , and other retrotransposon - like features including both a retroviral - like hypothetical gene and an rvt2-domain reverse transcriptase pseudogene . 
 the central dna transposase gene encodes , in eight exons , a predicted 160-kda protein producing blast alignments with en / spm - type transposons . except for a stop signal , 
 another orf encodes a ty1-copia - like reverse transcriptase with amino acid sequence domain yvddiil . 
 outside apparent ltrs , an 8-mer nucleotide sequence motif cactataa , near or within inverted repeat sequences , is hypothetical extreme termini . 
 a genome scan of arabidopsis thaliana found another example of a tnp2-domain transposase gene within an apparent ltr - retrotransposon on chromosome 4 . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS AND DISCUSSION
4. CONCLUSION