BACKGROUND
transposable elements are found in the genomes of almost all organisms, and are of ancient origin.
their ability to insert into new genomic locations makes them potent agents of phenotypic change, including various known pathologies  <cit> .
transposons are also found in parasites, for example the human enteric pathogen, entamoeba histolytica, a unicellular eukaryote contains three families of the autonomous non long terminal repeat  retrotransposon called ehline and its nonautonomous partner ehsine  <cit> .
the genome of the morphologically indistinguishable sibling species entamoeba dispar, , also contains three families of edlines and their partner edsines  <cit> .
it would be of interest to know whether these retrotransposons have any influence on pathogenesis-related gene expression.
lines and sines are known to influence the expression of neighboring genes by a variety of mechanisms, for example by providing alternative promoters, splicing and polyadenylation sites and by heterochromatinization  <cit> .
for this reason it is important to investigate whether these elements are located at syntenic positions in the e. histolytica and e. dispar genomes.
earlier investigations with a limited number of genomic loci showed that the sites occupied by ehsine <dig> in the e. histolytica genome were empty at homologous regions in e. dispar  <cit>  and conversely the sites occupied by edsine <dig> in the e. dispar genome were empty in e. histolytica  <cit> .
although an exhaustive genome-wide survey of lines and sines in entamoeba species has been reported  <cit> , a genome-wide comparison of the occupancy of these elements at syntenic loci in e. histolytica and e. dispar has not been carried out.
here we present results of a genome-wide comparison of ehsine <dig> and edsine <dig> in the two genomes.
in addition we address the question whether the differences in genomic locations of retrotransposons in these two organisms could be due to inherent differences in the retrotransposition machinery, particularly in the properties of the line-encoded endonuclease.
target primed reverse transcription is the mechanism by which non-ltr retrotransposons insert in the genome  <cit> .
since retrotransposition is initiated by the element-encoded endonuclease  making a nick at the bottom strand of the site of insertion, an important determinant of target site specificity could be the preferred nucleotide sequences recognized by the en.
we have earlier shown that the ehline1-encoded en  nicks preferentially at the consensus sequence 5'-gtatt-3', between a-t and t-t. here we have investigated whether the endonuclease domain encoded by edline <dig>  has a different target site specificity which could account for the lack of synteny in the location of sines in the two genomes.
methods
comparative analysis of e. histolytica and e. dispar with respect to the occupancy of sine <dig> elements
since the entamoeba genome has not yet been assembled completely for any species, we have done all of our comparative analysis on the basis of sine <dig> elements located on the scaffolds.
the genome sequence of e. histolytica having  <dig> scaffolds and e. dispar having  <dig> scaffolds was downloaded .
a total of  <dig> ehsine <dig> and  <dig> edsine <dig> elements having lengths greater than  <dig> bp were located.
the ehsine <dig> sequences were from huntley et al  <cit>  and the edsine <dig> sequences were from the feature table file of e. dispar .
for genes flanking the ehsine <dig> sequences the feature table file of e. histolytica  was used.
these were downloaded from ncbi and the genes upstream and downstream of sine <dig> elements in each of the species were located using perl coding.
finally the orthologues of these genes were searched in the other species using blast  <cit> .
the syntenic loci were checked to see whether sine <dig> was present there in both species.
to check the homology at scaffold level we used gata, a graphic alignment tool for comparative sequence analysis  <cit> .
syntenic loci were further examined to check for the presence of sine in both species.
dna sequence features of the sine <dig> insertion sites were computed by extracting flanking  <dig> bp sequences  from the respective genomic sites in e. histolytica and e. dispar, using perl scripting.
"dna scanner" was used as previously described  <cit>  to calculate the dna parameters, which include t rule, bendability, propeller twist, stacking energy, duplex stability, dna denaturation, protein-induced deformability, nucleosomal positioning and bending energy  <cit> .
construction and cloning of ed en
the consensus amino acid sequences of the e. dispar and e. histolytica endonuclease domains, ed en and eh en respectively were compared.
a total of  <dig> amino acid positions were different of which  <dig> amino acid residues were changed in the eh en sequence  <cit>  and  <dig> amino acid residues  were left unchanged to obtain the ed en sequence.
the desired mutations were introduced either by means of overlapping pcr  or by site-directed mutagenesis.
the primer pairs used are listed .
the ed en sequence thus obtained  was cloned into the ecori-noti site of pet <dig> vector  to yield the pet-ed-en construct.
expression and purification of recombinant ed en
this was done essentially as described for eh en  <cit> .
the pet-ed-en construct was transformed in escherichia coli bl- <dig> .
cells were grown in  <dig> ml of luria broth at 30°c to od <dig> of  <dig> .
for induction of hexa-his-tagged ed en, iptg was used to a final concentration of  <dig>  mm and the cells were further incubated for  <dig> hour.
the protein was purified by ni2+-nitrilotriacetic acid-agarose  affinity chromatography and eluted with  <dig> mm of imidazole.
the eluted fractions were checked for the protein by resolving on a 12% sds page; these fractions were pooled, dialyzed and stored at -20°c.
the enzyme activity of ed en was measured under the same conditions used for eh en, i.e.
ph  <dig> , at 37°c, and at mg2+ and nacl concentrations of  <dig> and  <dig> mm respectively  <cit> .
western blotting
the whole cell lysate of induced and uninduced bl- <dig>  cells was resolved on 12% sds page and transferred to pvdf membrane by semidry transfer method according to manufacturer's instructions .
the membrane was blocked overnight with 5% skimmed milk in pbs-t  and subsequently incubated with anti-his antibody or anti-eh en antibody for  <dig> hour followed by three times washing with pbs-t. the membrane was further incubated with horseradish peroxidase-conjugated secondary antibody and again washed with pbs-t thrice.
the protein was detected by the chemiluminescent hrp substrate .
nicking assay of radiolabeled  <dig> bp substrate
the  <dig> bp dna fragment was radiolabeled as described earlier  <cit> .
the labeled dna was resolved on 6% native gel, the band was visualized by ethidium bromide staining, excised from the gel and eluted by crush and soak method  <cit> .
the nicking assay was carried out as described for eh en and products were resolved by denaturing electrophoresis on 6-8% polyacrylamide gels containing  <dig> m urea  <cit> .
the gels were dried and autoradiographed in the phosphorimager .
nicking assay of pbs dna
supercoiled pbluescript  plasmid dna was purified by plasmid purification kit .
<dig> nm of purified ed en was incubated with 2- <dig> nm of supercoiled pbs dna.
the reaction was performed under the conditions employed for eh en  <cit> , at 37°c for  <dig> minutes and was stopped with  <dig> mm edta.
products were separated on  <dig> % agarose gel containing  <dig>  μg of ethidium bromide/ml.
under this condition the supercoiled dna migrated fastest, followed by linear and open circular form.
the intensity of supercoiled dna band was measured as a function of time, which gave the measure of the disappearance of supercoiled dna.
quantification was done by densitometry; the kinetic constants vmax, km and kcat were determined as described  <cit> .
the data obtained were the average of three independent determinations.
RESULTS
comparative analysis of ehsine1-containing regions of e. histolytica genome and syntenic regions of e. dispar
a total of  <dig> full-length sine <dig> elements  were identified in e. histolytica by genome sequence analysis.
syntenic regions corresponding to each of the ehsine1-containing loci were located in e. dispar.
to score for synteny entire scaffolds were matched in the two species using the program gata.
in 88% of cases where synteny was found, the syntenic regions matched throughout the scaffold, while in the rest synteny was not visible in some patches.
the presence or absence of any of the edsines  was determined at syntenic loci of e. dispar.
the results are summarized in figure 1a.
of the  <dig> ehsine1-containing loci of e. histolytica, syntenic regions in e. dispar could be predicted with certainty for  <dig> loci.
only these loci were included for further study - thus removing the contribution of differential sequence coverage in our comparative analysis.
loci not represented in both species due to differences in genome coverage, or difficulty in alignment, have not been included.
in addition, as stated above, the synteny stretched for the entire length of the scaffold.
of these  <dig> loci, sines were absent in e. dispar at  <dig> loci, were present at  <dig> loci and their presence or absence could not be determined at  <dig> loci.
amongst the  <dig> loci where sines were absent, no repeat element of any type was found at  <dig> loci , while line or edrc <dig> sequences were found at  <dig> loci.
amongst the  <dig> loci that contained a sine,  <dig> had edsine <dig> , one had a truncated copy of edsine <dig>  and five had edsine <dig> and/or edsine <dig>  amongst the  <dig> loci where presence or absence of sine could not be established, in  <dig> cases the scaffold ended within the locus in e. dispar, and in  <dig> cases the homology was restricted to genes on one side of the sine while there was no homology on the other side .
for  <dig> ehsine1-containing loci of e. histolytica, syntenic loci could not be found in e. dispar for the following reasons.
in many of these cases the scaffolds containing these loci were composed entirely of repeats , trna genes and repeats  or pseudogenes and repeats .
in  <dig> cases synteny could not be determined either because there were multiple copies of homologous genes, or homologous genes were located on multiple scaffolds, or there was a single gene in the scaffold.
comparative analysis of edsine1-containing regions of e. dispar genome and syntenic regions of e. histolytica
a total of  <dig> full-length sine <dig> elements  were identified in e. dispar by genome sequence analysis.
syntenic regions corresponding to each of the edsine1-containing loci were located in e. histolytica and the presence or absence of any of the ehsines  was determined as described above.
of the  <dig> loci, syntenic regions could be predicted with certainty for  <dig> loci .
of these, sines were absent in e. histolytica at  <dig> loci, were present at  <dig> loci and their presence or absence could not be determined at  <dig> loci.
amongst the  <dig> loci where sines were absent, no repeat element of any type was found at  <dig> loci , while line sequences were found at  <dig> loci.
amongst the  <dig> loci that contained a sine,  <dig> had ehsine <dig>  while  <dig> had ehsine <dig>  amongst the  <dig> loci where presence or absence of sine could not be established, in  <dig> cases the scaffold ended within the locus in e. histolytica, and in  <dig> cases the homology was restricted to genes on one side of the sine while there was no homology on the other side.
for  <dig> edsine1-containing loci, syntenic loci could not be found in e. histolytica for the reasons mentioned in the previous section.
in  <dig> cases the e. dispar loci were composed entirely of repeats while in  <dig> cases synteny could not be determined either because there were multiple copies of homologous genes, or homologous genes were located on multiple scaffolds, or there was a single gene in the scaffold.
sequence alignment of syntenic loci
from the above data it is clear that only in about 20% of cases where presence or absence of sine <dig> could be established at syntenic loci, are sine elements located in the same intergenic region  in both species.
in >80% of these loci sine <dig> was not found at the same location in both species.
since the elements in the two species have a common lineage and are closely related, what possible factors might account for these differences?
according to the target primed reverse transcription model, retrotransposition is initiated by the line-encoded endonuclease  nicking the bottom strand of the target site  <cit> .
hence it is reasonable to believe that the sequences preferentially nicked by the en could be the preferred insertion sites of the retrotransposon, and the behavior of en might influence the choice of target site of a non ltr retrotransposon.
since the eh en and ed en differ from each other at many amino acid positions , it is possible that the two enzymes may have evolved different recognition specificities.
to establish this we studied the properties of the edline1-encoded en and compared it with ehline1-encoded en.
cloning and expression of the edline <dig> endonuclease  polypeptide
to obtain the ed en coding sequence we used the eh en sequence  as a starting point.
ed en differs from eh en in  <dig> amino acid positions .
the eh en sequence was mutated in these positions  to obtain the ed en coding sequence.
this 782-bp ecori-not <dig> fragment was cloned in the e. coli expression vector pet30b.
the expressed protein contained his-tag, and together with other vector sequences at the amino terminus, it was  <dig> amino acids long, with an expected molecular mass of  <dig>  kda .
it was purified by nickel-agarose chromatography, and its identity was confirmed by using an anti-his tag antibody and anti-eh en antibody .
the ed en protein could nick a nonspecific substrate, pbs.
like the previously reported activity of eh en  <cit>  supercoiled pbs dna was efficiently nicked by the purified ed en protein to yield open circular and linear dnas.
the presence of discrete bands corresponding to open circular and linear forms shows that the enzyme makes predominantly single-strand nicks and not double-strand breaks .
kinetics of the ed en-catalyzed reaction with pbs supercoiled dna substrate under steady-state conditions
to determine the kinetics under steady-state conditions, reactions were carried out with the enzyme at a concentration of  <dig> nm and with pbs dna at a concentration of 2- <dig> nm .
the disappearance of supercoiled dna was determined by densitometric scanning as described for eh en  <cit> .
as mentioned in methods, all time-course results were the average of at least three independent determinations.
the variation observed at each time point was < <dig> % of the mean value .
although the variation in values of each data point was in the range of 4% in three replicates, the slopes for each set showed lesser variation .
kinetic parameters  were calculated from a lineweaver-burk plot .
km for pbs dna was calculated to be  <dig>  ±  <dig>  × 10- <dig> m. the catalytic constant, kcat,  was determined to be  <dig>  ±  <dig>  × 10- <dig> sec- <dig>  these values were comparable to the km  and kcat  of eh en  <cit>  and the km was comparable with the low km values  of restriction endonucleases determined with different dna substrates under different conditions of buffer and temperature  <cit> .
furthermore, the turnover number  of the enzyme was in the lower range of that reported for restriction endonucleases   <cit> .
the low turnover number of a retrotransposon-encoded endonuclease may have a significant role in limiting the rate of retrotransposition events in the genome.
from the above data we infer that the kinetic parameters of ed en are not significantly different from eh en.
nicking site sequence preference of ed en
we had earlier shown that eh en preferentially nicked a  <dig> bp fragment from e. histolytica precisely at the site where a sine <dig> element was known to insert in this region of the genome  <cit> .
to test whether ed en had a similar sequence preference as eh en, the same  <dig> bp fragment was incubated with ed en.
the nicking pattern, determined for the bottom strand, was exactly the same as that obtained with eh en.
three nicking hot spots were obtained, of which site # <dig> corresponded with the exact site of insertion of eh sine <dig> in vivo .
the sequences important for target site recognition, as determined for eh en  <cit> , were tested for ed en by altering the sequences immediately surrounding the nicked site # <dig>  transition mutations were introduced using oligonucleotides with the appropriately altered sequence to pcr amplify a  <dig> bp fragment from the  <dig> bp template .
the dna sequences thus obtained contained a normal site # <dig> and a mutated site # <dig>  the activity of ed en on the mutated site # <dig> was quantitated using site # <dig> as an internal control.
the results showed that changing the gg nucleotides  to tt decreased the activity to 10% for ed en  and changing the t nucleotide upstream of the nick to c increased the activity to 183% for ed en  .
these results show that both ed en and eh en are very similar in their target site specificity.
since the endonuclease domain of orf <dig> was used in these studies, the possibility of a complete orf <dig> protein displaying a different specificity in vivo cannot, however, be entirely ruled out.
dna structural features of e. dispar and e. histolytica sine <dig> insertion sites
we have earlier shown that sequence-dependent dna structural features may play an important role in site selection by sine elements in e. histolytica  <cit> .
here we have analyzed the same features in e. dispar to see if the sine insertion sites share these properties with e. histolytica.
the parameters checked are listed in 'methods'.
in general majority of the features showed similar pattern in both the species except for dna denaturation energy and free energy profile.
our previous study had shown that the insertion sites in e. histolytica are t-enriched and the content profile showed a significant peak at - <dig> bp relative to the insertion site.
similar profile was also observed for e. dispar with a peak at - <dig> bp .
statistical analysis by mann-whitney test on the difference of the average t content of insertion sites for e. histolytica and e. dispar suggested that there is no significant difference between the two.
similar results were obtained when other dna-based structural parameters were determined.
thus the sine1-occupied sites in e. histolytica and e. dispar share the same structural features.
we checked whether the intergenic regions at loci where sines were found in both genomes shared greater sequence similarity compared with loci where sines did not occur in both genomes.
however this was not found to be the case.
intergenic regions in both sets of loci showed overall sequence similarity in the range of 75-90%.
e. histolytica and e. dispar are very closely related sibling species  <cit>  which were in fact classified as a single species until they were re-described as two separate species  <cit> .
their close relationship is also evident from phylogeny based on line-derived rt sequences.
this analysis showed that all three families of lines and sines already existed in the common ancestor before e. histolytica and e. dispar separated into two distinct species  <cit> .
the great similarity between the two ens of e. histolytica and e. dispar, as found in this study, again shows that the basic retrotransposition machinery is highly conserved in these sibling species.
it is therefore possible that sines may indeed have occupied all of the potential insertion sites in the genome of the common ancestor of e. histolytica and e. dispar but many of the inserted elements may have been preferentially lost in each genome as the two species diverged from each other.
indeed the differential loss of retrotransposons from specific loci might have contributed to speciation  <cit> .
on the other hand it may be possible that sine expansion took place after the divergence of the two species, and only a sub set of the potential insertion sites in the e. histolytica and e. dispar genomes are currently occupied.
in that case one may expect that each of these extant genomes may possess a large number of 'empty' sites where sines could potentially insert in future.
a hallmark of retrotransposition is the appearance of target site duplication  following the insertion of a new element.
in syntenic loci an unoccupied site is expected to have one copy of the tsd which is duplicated in the occupied site.
we checked for tsd sequences in e. dispar unoccupied sites corresponding to the syntenic e. histolytica occupied sites.
we randomly picked  <dig> loci of e. histolytica, where sine <dig> is absent in e. dispar and looked for matches with the tsd at each locus.
at  <dig> loci we found very good match with the tsd sequence .
the occurrence of close matches of tsd sequences in the syntenic loci of e. dispar suggests that potential empty sites may exist where future retrotransposition events could take place.
CONCLUSIONS
our data show that the line-encoded endonucleases, eh en and ed en are essentially very similar, both in their kinetic properties and in their substrate sequence specificity.
the dna structural features of sine-occupied sites in e. histolytica and e. dispar are also similar.
however the elements do not insert at the same sites in the two species.
even in the 20% cases where the elements are located in the same intergenic regions in the two genomes the exact point of insertion is not the same.
it is possible that, despite the eh en and ed en being very similar, the complete retrotransposition machinery consisting of the ribonucleoprotein assembly of line-encoded orf <dig>  orf <dig> and the sine transcript might function differently in the two species, thus leading to the observed differences.
since the elements are very closely related, these differences are likely to be the result of subtle changes that got established after the divergence of the two species.
an experimental test of these functional changes requires the complete assembly of orf <dig> and orf <dig> of edline <dig>  on the other hand the possibility exists that the retrotransposition machineries in the two species are in fact identical and the observed differences are due to the stochastic nature of the insertion process.
the number of potential insertion sites of these elements is likely to be large since they do not insert at specific sequences.
the only known specificity of the process is that the elements in both e. histolytica and e. dispar insert only in intergenic regions and not within genes.
within intergenic regions they insert near t-rich stretches  <cit> .
as discussed above, in this scenario one would expect to find a large number of 'empty' sites in each genome which could be targets of future insertions.
this needs to be experimentally tested.
a further possibility is that common insertions did indeed occur in the two genomes, but these were subsequently lost in a differential manner due to selection pressure.
if the loss of sine copies from specific locations conferred a growth advantage to either species, these elements could well have shaped the physiological evolution of the two species, including their virulence properties.
whatever may have been the mechanisms and processes that determined the positioning of sines, the resultant effect is that sines occupy different intergenic locations in the two genomes.
it is well recognized that lines and sines can modulate the expression of genes in their vicinity by providing alternative promoters, splicing and polyadenylation sites and by heterochromatinization  <cit> .
the absence of sine <dig> in >80% of syntenic loci in the extant genomes of e. histolytica and e. dispar could result in differential expression of genes at these loci.
this could profoundly influence the phenotype of the two species, which needs to be explored.
recently lorenzi et al.
<cit>  in their reannotation and analysis of the e. histolytica genome have listed a large number of protein families showing high association with repetitive elements.
though the top three families which are associated with tes 100% of the time are hypothetical proteins, important known protein families are also listed.
these include gal/gal nac lectin, hsp <dig> bspa-like surface protein family and aig family associated with resistance to bacteria.
further analysis of a similar nature with e. dispar genome will give interesting information on the possible contribution of tes in regulating the expression of important genes that may influence pathogenesis.
authors' contributions
sb proposed and designed the research, drafted the final version of the manuscript, ab designed and analyzed the computational work.
vk performed the experiments regarding cloning, expression, purification and assay of ed en.
rs performed the computational work.
vpy helped in performing the enzyme kinetics of ed en, akg helped in the cloning of ed en.
all authors have participated in preparing the manuscript.
all authors have read and approved the final manuscript.
supplementary material
additional file 1
figure s <dig>  schematic representation of overlapping pcrs for construction of ed en.
sixteen sets of primers incorporating the desired mutations were designed.
pcr was done using pet-eh-en construct as template.
the  <dig> bp fragment containing ed en domain was cloned in pet <dig> vector at the ecori-noti site.
click here for file
additional file 2
table s <dig>  primer sets used for the construction of ed en.
click here for file
additional file 3
figure s <dig>  graphical representation of the syntenic region where sine <dig> is present in e. histolytica but absent in e. dispar.
for color code and arrows refer to figure s1-s17
click here for file
additional file 4
figure s3-s <dig>  graphical representation of the syntenic region where sine <dig> is present in both e. histolytica and e. dispar.
top and bottom green lines are showing the genomes of e. histolytica and e. dispar respectively.
red arrows show the eh and ed genes, and the gene orientation.
shaded black or red  lines show the identical region within e. histolytica and e. dispar.
darkness of the shade is proportional to the % identity.
blue arrows show the repeat elements.
sine <dig> has been shown above the marked yellow dot in both e. hitolytica and e. dispar in the syntenic region.
click here for file
additional file 5
figure s <dig>  graphical representation of syntenic region where sine <dig> is present in e. dispar but absent in e. histolytica.
for color code and arrows refer to figure s1-s <dig>
click here for file
acknowledgements
this work was supported by a grant to sb from indian council of medical research and by fellowship from council of scientific and industrial research, india to vk and akg.

 transposable elements are found in the genomes of almost all organisms, and are of ancient origin. their ability to insert into new genomic locations makes them potent agents of phenotypic change, including various known pathologies. transposons are also found in parasites, for example the human enteric pathogen, entamoeba histolytica, a unicellular eukaryote contains three families of the autonomous non long terminal repeat retrotransposon called ehline and its nonautonomous partner ehsine. the genome of the morphologically indistinguishable sibling species entamoeba dispar,, also contains three families of edlines and their partner edsines. it would be of interest to know whether these retrotransposons have any influence on pathogenesis-related gene expression. lines and sines are known to influence the expression of neighboring genes by a variety of mechanisms, for example by providing alternative promoters, splicing and polyadenylation sites and by heterochromatinization. for this reason it is important to investigate whether these elements are located at syntenic positions in the e. histolytica and e. dispar genomes. earlier investigations with a limited number of genomic loci showed that the sites occupied by ehsine in the e. histolytica genome were empty at homologous regions in e. dispar and conversely the sites occupied by edsine in the e. dispar genome were empty in e. histolytica. although an exhaustive genome-wide survey of lines and sines in entamoeba species has been reported, a genome-wide comparison of the occupancy of these elements at syntenic loci in e. histolytica and e. dispar has not been carried out. here we present results of a genome-wide comparison of ehsine and edsine in the two genomes. in addition we address the question whether the differences in genomic locations of retrotransposons in these two organisms could be due to inherent differences in the retrotransposition machinery, particularly in the properties of the line-encoded endonuclease. target primed reverse transcription is the mechanism by which non-ltr retrotransposons insert in the genome. since retrotransposition is initiated by the element-encoded endonuclease making a nick at the bottom strand of the site of insertion, an important determinant of target site specificity could be the preferred nucleotide sequences recognized by the en. we have earlier shown that the ehline1-encoded en nicks preferentially at the consensus sequence 5'-gtatt-3', between a-t and t-t. here we have investigated whether the endonuclease domain encoded by edline has a different target site specificity which could account for the lack of synteny in the location of sines in the two genomes. methods comparative analysis of e. histolytica and e. dispar with respect to the occupancy of sine elements since the entamoeba genome has not yet been assembled completely for any species, we have done all of our comparative analysis on the basis of sine elements located on the scaffolds. the genome sequence of e. histolytica having scaffolds and e. dispar having scaffolds was downloaded. a total of ehsine and edsine elements having lengths greater than bp were located. the ehsine sequences were from huntley et al and the edsine sequences were from the feature table file of e. dispar. for genes flanking the ehsine sequences the feature table file of e. histolytica was used. these were downloaded from ncbi and the genes upstream and downstream of sine elements in each of the species were located using perl coding. finally the orthologues of these genes were searched in the other species using blast. the syntenic loci were checked to see whether sine was present there in both species. to check the homology at scaffold level we used gata, a graphic alignment tool for comparative sequence analysis. syntenic loci were further examined to check for the presence of sine in both species. dna sequence features of the sine insertion sites were computed by extracting flanking bp sequences from the respective genomic sites in e. histolytica and e. dispar, using perl scripting. "dna scanner" was used as previously described to calculate the dna parameters, which include t rule, bendability, propeller twist, stacking energy, duplex stability, dna denaturation, protein-induced deformability, nucleosomal positioning and bending energy. construction and cloning of ed en the consensus amino acid sequences of the e. dispar and e. histolytica endonuclease domains, ed en and eh en respectively were compared. a total of amino acid positions were different of which amino acid residues were changed in the eh en sequence and amino acid residues were left unchanged to obtain the ed en sequence. the desired mutations were introduced either by means of overlapping pcr or by site-directed mutagenesis. the primer pairs used are listed. the ed en sequence thus obtained was cloned into the ecori-noti site of pet vector to yield the pet-ed-en construct. expression and purification of recombinant ed en this was done essentially as described for eh en. the pet-ed-en construct was transformed in escherichia coli bl-. cells were grown in ml of luria broth at 30°c to od of. for induction of hexa-his-tagged ed en, iptg was used to a final concentration of mm and the cells were further incubated for hour. the protein was purified by ni2+-nitrilotriacetic acid-agarose affinity chromatography and eluted with mm of imidazole. the eluted fractions were checked for the protein by resolving on a 12% sds page; these fractions were pooled, dialyzed and stored at -20°c. the enzyme activity of ed en was measured under the same conditions used for eh en, i.e. ph, at 37°c, and at mg2+ and nacl concentrations of and mm respectively. western blotting the whole cell lysate of induced and uninduced bl- cells was resolved on 12% sds page and transferred to pvdf membrane by semidry transfer method according to manufacturer's instructions. the membrane was blocked overnight with 5% skimmed milk in pbs-t and subsequently incubated with anti-his antibody or anti-eh en antibody for hour followed by three times washing with pbs-t. the membrane was further incubated with horseradish peroxidase-conjugated secondary antibody and again washed with pbs-t thrice. the protein was detected by the chemiluminescent hrp substrate. nicking assay of radiolabeled bp substrate the bp dna fragment was radiolabeled as described earlier. the labeled dna was resolved on 6% native gel, the band was visualized by ethidium bromide staining, excised from the gel and eluted by crush and soak method. the nicking assay was carried out as described for eh en and products were resolved by denaturing electrophoresis on 6-8% polyacrylamide gels containing m urea. the gels were dried and autoradiographed in the phosphorimager. nicking assay of pbs dna supercoiled pbluescript plasmid dna was purified by plasmid purification kit. nm of purified ed en was incubated with 2- nm of supercoiled pbs dna. the reaction was performed under the conditions employed for eh en, at 37°c for minutes and was stopped with mm edta. products were separated on % agarose gel containing μg of ethidium bromide/ml. under this condition the supercoiled dna migrated fastest, followed by linear and open circular form. the intensity of supercoiled dna band was measured as a function of time, which gave the measure of the disappearance of supercoiled dna. quantification was done by densitometry; the kinetic constants vmax, km and kcat were determined as described. the data obtained were the average of three independent determinations.  comparative analysis of ehsine1-containing regions of e. histolytica genome and syntenic regions of e. dispar a total of full-length sine elements were identified in e. histolytica by genome sequence analysis. syntenic regions corresponding to each of the ehsine1-containing loci were located in e. dispar. to score for synteny entire scaffolds were matched in the two species using the program gata. in 88% of cases where synteny was found, the syntenic regions matched throughout the scaffold, while in the rest synteny was not visible in some patches. the presence or absence of any of the edsines was determined at syntenic loci of e. dispar. the results are summarized in figure 1a. of the ehsine1-containing loci of e. histolytica, syntenic regions in e. dispar could be predicted with certainty for loci. only these loci were included for further study - thus removing the contribution of differential sequence coverage in our comparative analysis. loci not represented in both species due to differences in genome coverage, or difficulty in alignment, have not been included. in addition, as stated above, the synteny stretched for the entire length of the scaffold. of these loci, sines were absent in e. dispar at loci, were present at loci and their presence or absence could not be determined at loci. amongst the loci where sines were absent, no repeat element of any type was found at loci, while line or edrc sequences were found at loci. amongst the loci that contained a sine, had edsine, one had a truncated copy of edsine and five had edsine and/or edsine amongst the loci where presence or absence of sine could not be established, in cases the scaffold ended within the locus in e. dispar, and in cases the homology was restricted to genes on one side of the sine while there was no homology on the other side. for ehsine1-containing loci of e. histolytica, syntenic loci could not be found in e. dispar for the following reasons. in many of these cases the scaffolds containing these loci were composed entirely of repeats, trna genes and repeats or pseudogenes and repeats. in cases synteny could not be determined either because there were multiple copies of homologous genes, or homologous genes were located on multiple scaffolds, or there was a single gene in the scaffold. comparative analysis of edsine1-containing regions of e. dispar genome and syntenic regions of e. histolytica a total of full-length sine elements were identified in e. dispar by genome sequence analysis. syntenic regions corresponding to each of the edsine1-containing loci were located in e. histolytica and the presence or absence of any of the ehsines was determined as described above. of the loci, syntenic regions could be predicted with certainty for loci. of these, sines were absent in e. histolytica at loci, were present at loci and their presence or absence could not be determined at loci. amongst the loci where sines were absent, no repeat element of any type was found at loci, while line sequences were found at loci. amongst the loci that contained a sine, had ehsine while had ehsine amongst the loci where presence or absence of sine could not be established, in cases the scaffold ended within the locus in e. histolytica, and in cases the homology was restricted to genes on one side of the sine while there was no homology on the other side. for edsine1-containing loci, syntenic loci could not be found in e. histolytica for the reasons mentioned in the previous section. in cases the e. dispar loci were composed entirely of repeats while in cases synteny could not be determined either because there were multiple copies of homologous genes, or homologous genes were located on multiple scaffolds, or there was a single gene in the scaffold. sequence alignment of syntenic loci from the above data it is clear that only in about 20% of cases where presence or absence of sine could be established at syntenic loci, are sine elements located in the same intergenic region in both species. in >80% of these loci sine was not found at the same location in both species. since the elements in the two species have a common lineage and are closely related, what possible factors might account for these differences? according to the target primed reverse transcription model, retrotransposition is initiated by the line-encoded endonuclease nicking the bottom strand of the target site. hence it is reasonable to believe that the sequences preferentially nicked by the en could be the preferred insertion sites of the retrotransposon, and the behavior of en might influence the choice of target site of a non ltr retrotransposon. since the eh en and ed en differ from each other at many amino acid positions, it is possible that the two enzymes may have evolved different recognition specificities. to establish this we studied the properties of the edline1-encoded en and compared it with ehline1-encoded en. cloning and expression of the edline endonuclease polypeptide to obtain the ed en coding sequence we used the eh en sequence as a starting point. ed en differs from eh en in amino acid positions. the eh en sequence was mutated in these positions to obtain the ed en coding sequence. this 782-bp ecori-not fragment was cloned in the e. coli expression vector pet30b. the expressed protein contained his-tag, and together with other vector sequences at the amino terminus, it was amino acids long, with an expected molecular mass of kda. it was purified by nickel-agarose chromatography, and its identity was confirmed by using an anti-his tag antibody and anti-eh en antibody. the ed en protein could nick a nonspecific substrate, pbs. like the previously reported activity of eh en supercoiled pbs dna was efficiently nicked by the purified ed en protein to yield open circular and linear dnas. the presence of discrete bands corresponding to open circular and linear forms shows that the enzyme makes predominantly single-strand nicks and not double-strand breaks. kinetics of the ed en-catalyzed reaction with pbs supercoiled dna substrate under steady-state conditions to determine the kinetics under steady-state conditions, reactions were carried out with the enzyme at a concentration of nm and with pbs dna at a concentration of 2- nm. the disappearance of supercoiled dna was determined by densitometric scanning as described for eh en. as mentioned in methods, all time-course results were the average of at least three independent determinations. the variation observed at each time point was < % of the mean value. although the variation in values of each data point was in the range of 4% in three replicates, the slopes for each set showed lesser variation. kinetic parameters were calculated from a lineweaver-burk plot. km for pbs dna was calculated to be ± × 10- m. the catalytic constant, kcat, was determined to be ± × 10- sec- these values were comparable to the km and kcat of eh en and the km was comparable with the low km values of restriction endonucleases determined with different dna substrates under different conditions of buffer and temperature. furthermore, the turnover number of the enzyme was in the lower range of that reported for restriction endonucleases. the low turnover number of a retrotransposon-encoded endonuclease may have a significant role in limiting the rate of retrotransposition events in the genome. from the above data we infer that the kinetic parameters of ed en are not significantly different from eh en. nicking site sequence preference of ed en we had earlier shown that eh en preferentially nicked a bp fragment from e. histolytica precisely at the site where a sine element was known to insert in this region of the genome. to test whether ed en had a similar sequence preference as eh en, the same bp fragment was incubated with ed en. the nicking pattern, determined for the bottom strand, was exactly the same as that obtained with eh en. three nicking hot spots were obtained, of which site # corresponded with the exact site of insertion of eh sine in vivo. the sequences important for target site recognition, as determined for eh en, were tested for ed en by altering the sequences immediately surrounding the nicked site # transition mutations were introduced using oligonucleotides with the appropriately altered sequence to pcr amplify a bp fragment from the bp template. the dna sequences thus obtained contained a normal site # and a mutated site # the activity of ed en on the mutated site # was quantitated using site # as an internal control. the results showed that changing the gg nucleotides to tt decreased the activity to 10% for ed en and changing the t nucleotide upstream of the nick to c increased the activity to 183% for ed en. these results show that both ed en and eh en are very similar in their target site specificity. since the endonuclease domain of orf was used in these studies, the possibility of a complete orf protein displaying a different specificity in vivo cannot, however, be entirely ruled out. dna structural features of e. dispar and e. histolytica sine insertion sites we have earlier shown that sequence-dependent dna structural features may play an important role in site selection by sine elements in e. histolytica. here we have analyzed the same features in e. dispar to see if the sine insertion sites share these properties with e. histolytica. the parameters checked are listed in'methods'. in general majority of the features showed similar pattern in both the species except for dna denaturation energy and free energy profile. our previous study had shown that the insertion sites in e. histolytica are t-enriched and the content profile showed a significant peak at - bp relative to the insertion site. similar profile was also observed for e. dispar with a peak at - bp. statistical analysis by mann-whitney test on the difference of the average t content of insertion sites for e. histolytica and e. dispar suggested that there is no significant difference between the two. similar results were obtained when other dna-based structural parameters were determined. thus the sine1-occupied sites in e. histolytica and e. dispar share the same structural features. we checked whether the intergenic regions at loci where sines were found in both genomes shared greater sequence similarity compared with loci where sines did not occur in both genomes. however this was not found to be the case. intergenic regions in both sets of loci showed overall sequence similarity in the range of 75-90%. e. histolytica and e. dispar are very closely related sibling species which were in fact classified as a single species until they were re-described as two separate species. their close relationship is also evident from phylogeny based on line-derived rt sequences. this analysis showed that all three families of lines and sines already existed in the common ancestor before e. histolytica and e. dispar separated into two distinct species. the great similarity between the two ens of e. histolytica and e. dispar, as found in this study, again shows that the basic retrotransposition machinery is highly conserved in these sibling species. it is therefore possible that sines may indeed have occupied all of the potential insertion sites in the genome of the common ancestor of e. histolytica and e. dispar but many of the inserted elements may have been preferentially lost in each genome as the two species diverged from each other. indeed the differential loss of retrotransposons from specific loci might have contributed to speciation. on the other hand it may be possible that sine expansion took place after the divergence of the two species, and only a sub set of the potential insertion sites in the e. histolytica and e. dispar genomes are currently occupied. in that case one may expect that each of these extant genomes may possess a large number of'empty' sites where sines could potentially insert in future. a hallmark of retrotransposition is the appearance of target site duplication following the insertion of a new element. in syntenic loci an unoccupied site is expected to have one copy of the tsd which is duplicated in the occupied site. we checked for tsd sequences in e. dispar unoccupied sites corresponding to the syntenic e. histolytica occupied sites. we randomly picked loci of e. histolytica, where sine is absent in e. dispar and looked for matches with the tsd at each locus. at loci we found very good match with the tsd sequence. the occurrence of close matches of tsd sequences in the syntenic loci of e. dispar suggests that potential empty sites may exist where future retrotransposition events could take place.  our data show that the line-encoded endonucleases, eh en and ed en are essentially very similar, both in their kinetic properties and in their substrate sequence specificity. the dna structural features of sine-occupied sites in e. histolytica and e. dispar are also similar. however the elements do not insert at the same sites in the two species. even in the 20% cases where the elements are located in the same intergenic regions in the two genomes the exact point of insertion is not the same. it is possible that, despite the eh en and ed en being very similar, the complete retrotransposition machinery consisting of the ribonucleoprotein assembly of line-encoded orf orf and the sine transcript might function differently in the two species, thus leading to the observed differences. since the elements are very closely related, these differences are likely to be the result of subtle changes that got established after the divergence of the two species. an experimental test of these functional changes requires the complete assembly of orf and orf of edline on the other hand the possibility exists that the retrotransposition machineries in the two species are in fact identical and the observed differences are due to the stochastic nature of the insertion process. the number of potential insertion sites of these elements is likely to be large since they do not insert at specific sequences. the only known specificity of the process is that the elements in both e. histolytica and e. dispar insert only in intergenic regions and not within genes. within intergenic regions they insert near t-rich stretches. as discussed above, in this scenario one would expect to find a large number of'empty' sites in each genome which could be targets of future insertions. this needs to be experimentally tested. a further possibility is that common insertions did indeed occur in the two genomes, but these were subsequently lost in a differential manner due to selection pressure. if the loss of sine copies from specific locations conferred a growth advantage to either species, these elements could well have shaped the physiological evolution of the two species, including their virulence properties. whatever may have been the mechanisms and processes that determined the positioning of sines, the resultant effect is that sines occupy different intergenic locations in the two genomes. it is well recognized that lines and sines can modulate the expression of genes in their vicinity by providing alternative promoters, splicing and polyadenylation sites and by heterochromatinization. the absence of sine in >80% of syntenic loci in the extant genomes of e. histolytica and e. dispar could result in differential expression of genes at these loci. this could profoundly influence the phenotype of the two species, which needs to be explored. recently lorenzi et al. in their reannotation and analysis of the e. histolytica genome have listed a large number of protein families showing high association with repetitive elements. though the top three families which are associated with tes 100% of the time are hypothetical proteins, important known protein families are also listed. these include gal/gal nac lectin, hsp bspa-like surface protein family and aig family associated with resistance to bacteria. further analysis of a similar nature with e. dispar genome will give interesting information on the possible contribution of tes in regulating the expression of important genes that may influence pathogenesis. authors' contributions sb proposed and designed the research, drafted the final version of the manuscript, ab designed and analyzed the computational work. vk performed the experiments regarding cloning, expression, purification and assay of ed en. rs performed the computational work. vpy helped in performing the enzyme kinetics of ed en, akg helped in the cloning of ed en. all authors have participated in preparing the manuscript. all authors have read and approved the final manuscript. supplementary material additional file 1 figure s schematic representation of overlapping pcrs for construction of ed en. sixteen sets of primers incorporating the desired mutations were designed. pcr was done using pet-eh-en construct as template. the bp fragment containing ed en domain was cloned in pet vector at the ecori-noti site. click here for file additional file 2 table s primer sets used for the construction of ed en. click here for file additional file 3 figure s graphical representation of the syntenic region where sine is present in e. histolytica but absent in e. dispar. for color code and arrows refer to figure s1-s17 click here for file additional file 4 figure s3-s graphical representation of the syntenic region where sine is present in both e. histolytica and e. dispar. top and bottom green lines are showing the genomes of e. histolytica and e. dispar respectively. red arrows show the eh and ed genes, and the gene orientation. shaded black or red lines show the identical region within e. histolytica and e. dispar. darkness of the shade is proportional to the % identity. blue arrows show the repeat elements. sine has been shown above the marked yellow dot in both e. hitolytica and e. dispar in the syntenic region. click here for file additional file 5 figure s graphical representation of syntenic region where sine is present in e. dispar but absent in e. histolytica. for color code and arrows refer to figure s1-s click here for file acknowledgements this work was supported by a grant to sb from indian council of medical research and by fellowship from council of scientific and industrial research, india to vk and akg.
further we found that the physical properties of the dna sequences adjoining the insertion sites were similar in both species. entamoeba histolytica and entamoeba dispar are closely related protistan parasites but while e. histolytica can be invasive, e. dispar is completely non pathogenic. a genome-wide comparison of the location of these elements in the e. histolytica and e. dispar genomes has not been carried out. here we extracted all genomic occurrences of full-length copies of ehsine in the e. histolytica genome and matched them with the homologous regions in e. dispar, and vice versa, wherever it was possible to establish synteny. sines may indeed have occupied all of the insertion sites in the genome of the common ancestor of e. histolytica and e. dispar but these may have been subsequently lost from some locations. it is also not known whether the retrotransposition machinery works similarly in both species. our data shows that the basic retrotransposition machinery is conserved in these sibling species. the absence of sine in 80% of syntenic loci could affect the phenotype of the two species, including their pathogenic properties, which needs to be explored. we found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. alternatively, sine expansion took place after the divergence of the two species. we checked whether the different genomic location in the two species was due to differences in the activity of the line-encoded endonuclease which is required for nicking the target site.
 entamoeba histolytica and entamoeba dispar are closely related protistan parasites but while e. histolytica can be invasive, e. dispar is completely non pathogenic. transposable elements constitute a significant portion of the genome in these species; there being three families of lines and sines. these elements can profoundly influence the expression of neighboring genes. thus their genomic location can have important phenotypic consequences. a genome-wide comparison of the location of these elements in the e. histolytica and e. dispar genomes has not been carried out. it is also not known whether the retrotransposition machinery works similarly in both species. the present study was undertaken to address these issues.  here we extracted all genomic occurrences of full-length copies of ehsine in the e. histolytica genome and matched them with the homologous regions in e. dispar, and vice versa, wherever it was possible to establish synteny. we found that only about 20% of syntenic sites were occupied by sine in both species. we checked whether the different genomic location in the two species was due to differences in the activity of the line-encoded endonuclease which is required for nicking the target site. we found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. hence the differential distribution of sines in these species is not likely to be influenced by the endonuclease. further we found that the physical properties of the dna sequences adjoining the insertion sites were similar in both species.  our data shows that the basic retrotransposition machinery is conserved in these sibling species. sines may indeed have occupied all of the insertion sites in the genome of the common ancestor of e. histolytica and e. dispar but these may have been subsequently lost from some locations. alternatively, sine expansion took place after the divergence of the two species. the absence of sine in 80% of syntenic loci could affect the phenotype of the two species, including their pathogenic properties, which needs to be explored. 