BACKGROUND
mariner-like elements  are class ii transposons found in most eukaryotic genomes.
these elements are discrete dna fragments that are able to move around within eukaryotic genomes, and in some cases they make up a high proportion of these genomes.
mles consist of a dna fragment of  <dig> to  <dig> base pairs  that contains a single transposase-encoding gene without an intron.
this gene is flanked by two short inverted terminal repeats  of  <dig> to  <dig> bp in length.
the widespread occurrence of mles in animal genomes is considered to be attributable to the fact that their transposition does not require host factors since, in vitro, the transposase  encoded by these elements is all that is required to catalyze all the transposition steps  <cit> .
the requirements for mariner transposition seem to be very simple, involving just two itrs separated by a dna fragment, a tpase source, and magnesium cations as cofactors  <cit> .
however, several factors can impair the ability of mles to achieve their own transposition in vivo.
physical and biochemical conditions may interfere with the transposition efficiency.
it has previously been demonstrated that in vitro the himar <dig> tpase  is about twice as efficient at 28°c as at  <dig> or 40°c  <cit> .
in addition, data from in vitro and in vivo assays have shown that mle nucleic acid components are also interfering factors.
the quality of the itr sequence is crucial for the transposition efficiency of mos <dig>  <cit> , and the untranslated regions  located between the itr and the tpase orf at both ends of the transposon act as transposition enhancers  <cit> .
inner regions of the transposon also modulate transposition efficiency in vivo, since transposition depends on the size of the dna stuffer between the itrs  <cit> , and also in some cases to the quality of this inner sequence  <cit> .
so far, studies investigating factors that modulate mos <dig> transposition have been fragmentary  <cit> .
mos <dig> is a potential tool in gene transfer and mutagenesis, and so we think that it is important to investigate these factors in more detail.
in this study, we investigated the impact on the ability of mos <dig> to transpose of two factors:  the dna configuration of the transposon donor, and  the properties of the stuffer.
our main findings concern four points.
first, we show that the nucleic acid sequence of the dna stuffer , and its length, can markedly impair transposition efficiency.
second, our study provides the first in vitro experimental demonstration that mites of mle origin are able to transpose.
we therefore propose a model for the physical constraint associated with the size of these small transposons.
third, our studies show that excessive temperature down-regulates mos <dig> transposition, yielding excision products that are unable to re-integrate.
finally, we have demonstrated that super-helicity of the transposon donor has a dramatic impact on transposition efficiency.
RESULTS
impact of the stuffer size on mos <dig> transposition
it had already been pointed out that increasing the stuffer size alters the transposition efficiency for himar <dig>  <cit>  and mos <dig>  <cit> .
we re-investigated this feature for mos <dig> using 3tet <dig> transposons enlarged from the 3' end of the tetracycline marker in bacterial transposition assays.
our findings confirmed that increasing the stuffer size did indeed dramatically decrease the transposition efficiency .
the impact of decreasing stuffer size was also investigated using mos <dig> pseudo-transposons made with small marker genes encoding resistance to zeocin, blasticidin, and puromycin .
our findings show that 3bla <dig>  3zeo <dig> and 3puro <dig> transposons were able to transpose efficiently .
transposition of minute transposon
to transpose, the mos <dig> transposon has to be bent, bringing the itr together  <cit> .
dna fragments that are longer than the bending persistence length  are spontaneously bent, with little force.
in contrast, significant bending of dna fragments less than 90-bp in length requires considerable force  <cit> .
these constraints should prevent the transposition of mle with stuffer dna less than 90-bp in length: this looks in contradiction with the presence of thousands of 80-bp hsmar <dig> mle interspersed within the human genome  <cit> .
since no antibiotic marker less than 90-bp was available, we designed a novel transposition assay .
this assay was monitored in vitro, ensuring that the bending of the dna stuffer was not due to a host factor.
we were able to recover surviving dh5α bacteria, indicating that the 122-bp mos <dig> transposon was indeed able to transpose within the ccdb+ gene.
sequencing the ccdb+ gene indicated that five of the twenty clones analyzed corresponded to real transpositions of the 122-bp mos <dig> transposon, as shown by the duplication of a ta dinucleotide at the integration site .
the other clones are due to bacteria that naturally escape the toxin encoded by the ccdb+ gene, as indicated by the manufacturer .
these findings were not consistent with what we know about the bending ability of small, double-stranded dna fragments.
since there was no helping host factor in our in vitro assay, we searched for sources of flexibility that would allow the bending required for assembling the synaptic complex.
the binding of a protein to dna is often associated with a bend in the dna.
bending of itrs has been demonstrated for tn <dig>  tn <dig> and is <dig> bacterial transposons  <cit> .
we therefore first investigated whether mos <dig> bends its itr.
emsa were performed using six  <dig> bp fragments containing the  <dig> bp 3'itr or 5'itr at various positions  and purified mbp-mos <dig>  the mobility differences between the mos1-itr complexes formed with the six different probes indicated that the mos <dig> did bend the itr.
the possibility that these bends were due to single strand dna cleavage at the outer or the inner extremity of the itr was ruled out as all the experimental steps were carried out for  <dig> minutes at 4°c, conditions for which the cleavage activity of the tpase is inhibited.
fig.
2b shows a representative data obtained with probes containing the 5'itr.
similar data were obtained with probes containing the 3'itr.
the rf of all the observed complexes was measured and plotted versus the position of the center of each probe .
for each probe, two single end complexes  were obtained, as expected  <cit> .
the sinus curves calculated from our data allowed us to map the center of the mos1-induced bend, located at the lowest point of the curve.
this was at position  <dig>  for sec <dig>  which located the bending core around the 21st nucleotide of both itr .
for sec <dig>  the lowest point of the curve was around position  <dig> , which located the bending core around the 18th nucleotide for both itrs .
these values are the means  for at least five independent experiments.
zhou's equation  <cit>  was used to determine bending angles of  <dig> °  and  <dig> °  for the sec <dig> formed with the 3' itr- and the 5' itr-containing fragments respectively, and  <dig> °  and  <dig> °  for the sec <dig> formed on the 3' itr- and the 5' itr-containing fragments respectively.
in conclusion, our results indicated that both 3'- and 5'-itr were bent at angles of about 90° as a result of mos <dig> binding.
interestingly, these angles may facilitate the assembly of a transposition complex in a minute transposon, such as the-one investigated here, since the stuffer fragment needs only form an angle of less than 90° to bring the transposon ends together which is quite possible for a 66-bp dna segment  <cit> .
another source of dna flexibility, such as single strand dna cleavage at the inner end of the itr, was also investigated.
the cleavages produced by mos <dig> in sec <dig> and pec <dig> were therefore re-investigated, taking into account the inner part of the itr .
we have already published that the itr is mainly cleaved at the 3'-end of the transferred strand and at positions + <dig> and + <dig> on the non-transferred strand, although several other cleaved positions were detected in the flanking dna on both strands   <cit> .
analysis of the bottom part of the gel also reveals that a single strand dna cleavage occurred on the transferred strand at position  <dig>  which is located at the inner end of the itr .
impact of stuffer sequence on mos <dig> transposition
two questions were addressed concerning the impact on transposition efficiency of the gc content of the transposon sequence, and of the number of ta dinucleotides in the transposon sequence.
we first prepared nine pseudo-mos <dig> with gc contents ranging from  <dig>  to  <dig> %.
the reference construct was the pbc-3tet <dig>  which has a gc content of  <dig> %.
in bacterial transposition assays, the transposition efficiency of the ten pseudo-transposons ranged over two orders of magnitude .
the data were plotted, and the correlation between the two parameters  was tested using the spearman correlation test with a significance threshold of  <dig>  .
nine of the  <dig> constructs plotted in this analysis were included in the ellipse , and the correlation between gc content and transposition frequency had a coefficient of about  <dig> .
this coefficient was significant enough to consider that there was a correlation between gc content and transposition frequency.
however, the facts that one transposon construct fell outside the ellipse, and that two other transposon constructs with gc contents close to 56% differ by more than one order of magnitude indicates that gc content is not the only sequence parameter that affects the stuffer quality for transposition.
we then prepared two modified genes encoding for tetracycline resistance: the first was ta-rich , and the second was ta-poor , but this did not have any marked effect on the percentage of gc .
these two genes were used as markers in transposition assays in bacteria.
it was expected that reducing the ta density would result in increased transposition efficiency, simply because some suicide auto-integrations would be avoided.
we observed no correlation between the ta numbers , and the transposition frequency .
this point was confirmed by the fact that a bleomycin resistance gene without the ta dinucleotide  gave the same result in transposition assays in bacteria as the wild type bleomycin resistance gene .
impact of temperature on mos <dig> transposition efficiency
in vitro transposition assays were performed at four temperatures,  <dig>   <dig>   <dig> and 37°c .
our results are in agreement with those previously obtained for himar <dig>  <cit> , as we found an optimal temperature of about 28°c, with no significant differences found between 28°c and 30°c .
since we had previously shown that mos <dig> binds to its itr with similar efficiency regardless of temperature  <cit> , we therefore checked whether a temperature effect was observed for mos <dig> excision or not.
time course excision assays were performed either at  <dig> or 37°c, using pbc-3tet <dig> plasmid as the transposon donor, and purified mbp-mos <dig>  changing the excision temperature led to two main differences .
the first was that, at 37°c, a marked dna degradation occurred, leading to the loss of about 50% of the dna two hours into the reaction, whereas no dna loss was observed after  <dig> hours at 30°c.
the second difference was that, despite the dna degradation, the pseudo-mos <dig>  accumulated at 37°c, whereas it was not detectable at 30°c.
this is not very surprising because an accurately excised transposon tends to be reinserted rather than accumulating.
indeed, insertion products were seen in the assay performed at 30°c, but not in the assay performed at 37°c .
this was in total agreement with the fact that more efficient transposition was observed at 30°c than at 37°c.
the dna degradation and the 3tet <dig> accumulation detected at 37°c might be due to a single phenomenon: at 37°c, mos <dig> cleaves dna not only at the end of the itr but anywhere in the dna.
this loss of specificity may promote the production of excised 3tet <dig> with extremities that are unable to integrate.
organization of excised pseudo-mos <dig> produced at 37°c
the organization of the pseudo-mos <dig> that accumulated at 37°c was analyzed using a variant of the pbc3tet <dig>  pbc-3kana <dig>  a transposon donor with a unique clai site in 3kana <dig> .
in southern blot analyses, pbc-3kana <dig> is detected both as open circle and linear molecules, whereas the excised 3kana <dig> accumulates as a double-band of  <dig>  kbp .
due to the sensibility of the technique, integration events are also detected.
the detection of a double-band of about  <dig> -kpb  suggests that excision products could exist both in linear and circular configurations.
southern analysis of the dna samples digested by clai was then monitored.
once cleaved by clai, the band with the highest apparent molecular weight in undigested dna samples was shortened accordingly to the position of the restriction clai site .
this suggests that this band corresponds to a linear excision product.
in contrast, the band of lower intensity in undigested dna samples is unaffected or only slightly by clai digestion, thus suggesting that it corresponds to a circular excision product or that it had no clai site.
primer name
the excision products were therefore purified by agarose gel elution from untreated dna samples.
they were submitted to pcr using primers anchored in an inverse orientation in the sequence of the kanamycin resistance gene, kanajunc <dig> and kanajunc <dig> , in order to amplify itr junctions looking like those isolated from bombyx mori cells transformed by mos1-based vectors  <cit> .
the amplification of a fragment of about 950-bp is expected if the circular forms correspond to the circularization of an excised full-length transposon .
fragments ranging from  <dig> to 800-bp were obtained and cloned.
twelve were sequenced: they all contained the clai site, and a single itr, inserted into or close to the kanamycin resistance gene .
this fact, combined with the fact that they were shorter than expected, indicates that these fragments resulted from intra-molecular integration events.
among the twelve fragments analyzed here, four came from auto-integration events occurring in a dinucleotide that was not a ta but tt , tc , tg  or ca  dinucleotides.
in conclusion, our data are consistent with the fact that some of mos <dig> excision products formed at 37°c are not able to perform integration into a target dna.
this is due to the formation of  linear products with inefficient extremities and  circular products resulting from intra-molecular auto-integration.
taken together, these events account for the decreased transposition efficiency observed at 37°c.
impact of the transposon donor configuration on mos <dig> transposition
in vitro transpositions were performed using a linear plasmid donor of transposon  and compared to those obtained using a super coiled transposon donor .
linearization of the transposon source dramatically reduced the transposition efficiency  at 28°c, and hindered transposition at other temperatures.
excision efficiencies of a super-helical donor were then compared to those obtained with the same donor linearized by ncoi.
when the super-helical pbc-3tet <dig> was used as the substrate, products were digested by ncoi at the end of the experiment, making it possible to carry out direct comparisons.
excision assays have been first analyzed by tracing the dead-end products of the reaction, i.e.
bands α and δ. our data therefore show no major difference between the two reactions.
analyses of the time taken to obtain 50% of α and δ fragments  indicated that it was about  <dig> and  <dig> hours for the super-coiled and linear donors, respectively .
furthermore, the curves differ in shape, the production of α + δ  being faster with the circular substrate.
this suggested differences between the two excision reactions.
this difference was studied measuring the patterns of appearance of the β and γ fragments that are not dead-end products of the reaction.
indeed, when mos <dig> excision occurs within the synaptic complex, β and γ are not expected to accumulate, because cleavage is believed to occur in a concerted fashion at both itrs.
much more β and γ accumulated when the donor was linear, rising to a peak before decreasing, as the result of cleaving the second itr .
in contrast, little β and γ accumulated when the substrate was super coiled, reaching a maximum of 10-fmoles.
these data supported the idea that the concerted cleavage at both itrs is reduced or abolished when the substrate is linear, a phenomenon that may account for an overall decrease in the transposition efficiency.
finally, transposition assays presented in fig.
5a corresponded to  <dig> minutes reactions.
at this time, excision and re-integration of the transposon from the circular substrate had already occurred  whereas this had not occurred for the linear substrate .
data in this section indicates that the excision is probably one of the limiting factors that accounts for the drastic decrease in transposition efficiency when the donor is linear.
at least two non-mutually exclusive explanations can be proposed:  excision from a linear donor is slower and  excision from a linear donor takes place independently at both itrs .
discussion
in addition to requiring itr and the tpase, previous studies have demonstrated that the transposition of mle or tle requires critical parameters that interfere with transposition efficiency.
these parameters include  tpase concentrations, demonstrated in vitro for himar <dig>  <cit>  and mos <dig>  <cit>  and in vivo for mos <dig>  <cit> ,  temperature  <cit> ,  size of the stuffer, demonstrated in vitro for tc <dig>  <cit> ,  sequence of the stuffer, demonstrated in vivo for mos <dig>  <cit> ,  structural configuration of the target dna, demonstrated in vitro for tc <dig>  <cit> .
in this study, we have used an in vitro approach to address several parameters affecting mos <dig> transposition.
parameters for optimal mariner transposition
our results highlight the importance of the stuffer sequence since they show that the gc content is an important factor, even if it is probably not the only parameter that affects the stuffer performance for transposition.
in silicio analyses  failed to establish a correlation between dna curvature and/or flexibility and transposition efficiency.
our results also confirm the impact of stuffer length on transposition efficiency, as already shown  <cit> .
length dependence of mos <dig> transposition might rely on auto-integration after excision.
this is supported by the fact that large sophisticated transposons such as the phage mu or tn <dig> have specific mechanisms that prevent the transposon from inserting into itself  <cit> .
in contrast, simple elements such as mariners lack this function.
a third parameter that controls mos <dig> transposition rate is the temperature.
interestingly, elevated temperature  promotes auto-integration events.
auto-integration does not absolutely require a ta dinucleotide.
this fact, associated with the fact that the percentage of ta dinucleotides does not influence mos <dig> transposition efficiency, rules out the possibility of designing large transgenes containing no  ta to limit the length-dependence of mos <dig> transposition.
mariner excision: making a molecule that produces efficient integration
data presented here highlight an important, although not new, idea: transposition efficiency is in great part controlled by the quality of the excision product.
this is in contradiction with published data  <cit>  that support the idea that cleavages at mles ends are far from accurate .
however, all these data were collected in vitro, using linear, double-stranded oligonucleotides.
on the other hand, mle in vivo excision footprints are often less variable, and closely match what the model predicts, i.e.
hydrolysis at the 3' end of the element, and 5' break two or three nucleotides within the transposon  <cit> .
this suggests that experimental conditions can bias the description of excision product ends.
our data allow proposing that there is a relationship between the super-helicity of the donor dna, and the production of efficient excision products, through the assembly of the synaptic complexes.
indeed, mos <dig> excision from a super-coiled donor yields transposition that is 1000-fold more efficient than that resulting from a linear donor plasmid .
these findings are fully consistent with super-helicity acting as a regulatory factor of mariner transposition in vitro, and probably in vivo.
from this standpoint, mariner looks like many other transposons.
for instance, mu transposition requires dna super coiling in the donor dna  <cit>  whereas tn <dig> needs either dna super coiling in the dna substrate, or ihf acting as a "super coiling relief factor"  <cit> .
super coiling is probably not required for the chemical steps of the transposition, but might be a limiting factor for the assembly of mariner transposition complexes.
although mariner transposition has been shown to take place in vitro, we cannot rule out the possibility that host factors may assist the transposase during in vivo transposition.
mite transposition
mites are small, repetitive, dna elements ranging in size from approximately  <dig> to  <dig> bp that are interspersed within eukaryotic genomes.
their sequence is palindromic, at-rich, and has no coding capacity.
for their mobility, they generally use a source of tpase in trans, originating from a related complete transposon copy  <cit> .
our data indicate that the size of mites of mle origin does not limit their ability to transpose.
for the smallest ones, found in the human genome  or in that of the ant messor bouvieri , the binding of mos <dig> causes major itrs bending, thus bringing the two ends of the transposon close to each other.
in addition, the inner itr cleavage by mos <dig> could also allows the itr bending.
similar cleavage was found on the non-transferred strand of the hsmar <dig> itr cleaved by hsmar <dig>  no host factor is therefore required to bend the dna and assemble the synaptic complex .
this model provides a very satisfying explanation of the mobility of the mle mites, because it explains how a minute mos <dig> can assemble a functional synaptic complex similar to that of the full-length mle.
moreover, it is supported by in vitro transposition data.
however, it does not explain why most of the mites, including those derived from mle, display a palindromic organization throughout their sequence.
in contrast to what was previously proposed  <cit> , our data suggest that complete palindromy is not a requirement for mite mobility, and that it could therefore result from other constraints.
CONCLUSIONS
non-viral vectors are presently invading the world of gene transfer and mutagenesis.
among the tools that are currently under evaluation, the transposon mos <dig> raises a growing interest.
in fact, it is perceived as a small versatile and quasi-universal means of transforming a variety of cells, except mammal ones  <cit> .
however, as for any new tool, it is necessary to control every parameter driving its transposition, and, in a first place, the factors that might limit its efficiency and specificity for dna transfer.
in the present work, we have investigated several parameters, particularly those related to the structure of the transposon.
in this respect, we have demonstrated that the dna sequence  as well as the length of the inner part of the transposon have a crucial effect on the transposition efficiency.
our results provide, for the first time, an experimental in vitro demonstration that mites of mle origin that are as small as 80– <dig> bp are able to transpose.
hence, we propose a model that bypasses the physical constraints associated to the transposition of such small transposons.
we have also demonstrated that the super-helicity of the transposon impacts considerably on the efficiency of transposition.
finally, we have found that mariner transposition is dependent on the temperature.
the fact that higher temperature disfavors re-integration of the excision products might be related to the transposase enzymatic properties.

 mariner-like elements are class ii transposons found in most eukaryotic genomes. these elements are discrete dna fragments that are able to move around within eukaryotic genomes, and in some cases they make up a high proportion of these genomes. mles consist of a dna fragment of to base pairs that contains a single transposase-encoding gene without an intron. this gene is flanked by two short inverted terminal repeats of to bp in length. the widespread occurrence of mles in animal genomes is considered to be attributable to the fact that their transposition does not require host factors since, in vitro, the transposase encoded by these elements is all that is required to catalyze all the transposition steps. the requirements for mariner transposition seem to be very simple, involving just two itrs separated by a dna fragment, a tpase source, and magnesium cations as cofactors. however, several factors can impair the ability of mles to achieve their own transposition in vivo. physical and biochemical conditions may interfere with the transposition efficiency. it has previously been demonstrated that in vitro the himar tpase is about twice as efficient at 28°c as at or 40°c. in addition, data from in vitro and in vivo assays have shown that mle nucleic acid components are also interfering factors. the quality of the itr sequence is crucial for the transposition efficiency of mos, and the untranslated regions located between the itr and the tpase orf at both ends of the transposon act as transposition enhancers. inner regions of the transposon also modulate transposition efficiency in vivo, since transposition depends on the size of the dna stuffer between the itrs, and also in some cases to the quality of this inner sequence. so far, studies investigating factors that modulate mos transposition have been fragmentary. mos is a potential tool in gene transfer and mutagenesis, and so we think that it is important to investigate these factors in more detail. in this study, we investigated the impact on the ability of mos to transpose of two factors: the dna configuration of the transposon donor, and the properties of the stuffer. our main findings concern four points. first, we show that the nucleic acid sequence of the dna stuffer, and its length, can markedly impair transposition efficiency. second, our study provides the first in vitro experimental demonstration that mites of mle origin are able to transpose. we therefore propose a model for the physical constraint associated with the size of these small transposons. third, our studies show that excessive temperature down-regulates mos transposition, yielding excision products that are unable to re-integrate. finally, we have demonstrated that super-helicity of the transposon donor has a dramatic impact on transposition efficiency.  impact of the stuffer size on mos transposition it had already been pointed out that increasing the stuffer size alters the transposition efficiency for himar and mos. we re-investigated this feature for mos using 3tet transposons enlarged from the 3' end of the tetracycline marker in bacterial transposition assays. our findings confirmed that increasing the stuffer size did indeed dramatically decrease the transposition efficiency. the impact of decreasing stuffer size was also investigated using mos pseudo-transposons made with small marker genes encoding resistance to zeocin, blasticidin, and puromycin. our findings show that 3bla 3zeo and 3puro transposons were able to transpose efficiently. transposition of minute transposon to transpose, the mos transposon has to be bent, bringing the itr together. dna fragments that are longer than the bending persistence length are spontaneously bent, with little force. in contrast, significant bending of dna fragments less than 90-bp in length requires considerable force. these constraints should prevent the transposition of mle with stuffer dna less than 90-bp in length: this looks in contradiction with the presence of thousands of 80-bp hsmar mle interspersed within the human genome. since no antibiotic marker less than 90-bp was available, we designed a novel transposition assay. this assay was monitored in vitro, ensuring that the bending of the dna stuffer was not due to a host factor. we were able to recover surviving dh5α bacteria, indicating that the 122-bp mos transposon was indeed able to transpose within the ccdb+ gene. sequencing the ccdb+ gene indicated that five of the twenty clones analyzed corresponded to real transpositions of the 122-bp mos transposon, as shown by the duplication of a ta dinucleotide at the integration site. the other clones are due to bacteria that naturally escape the toxin encoded by the ccdb+ gene, as indicated by the manufacturer. these findings were not consistent with what we know about the bending ability of small, double-stranded dna fragments. since there was no helping host factor in our in vitro assay, we searched for sources of flexibility that would allow the bending required for assembling the synaptic complex. the binding of a protein to dna is often associated with a bend in the dna. bending of itrs has been demonstrated for tn tn and is bacterial transposons. we therefore first investigated whether mos bends its itr. emsa were performed using six bp fragments containing the bp 3'itr or 5'itr at various positions and purified mbp-mos the mobility differences between the mos1-itr complexes formed with the six different probes indicated that the mos did bend the itr. the possibility that these bends were due to single strand dna cleavage at the outer or the inner extremity of the itr was ruled out as all the experimental steps were carried out for minutes at 4°c, conditions for which the cleavage activity of the tpase is inhibited. fig. 2b shows a representative data obtained with probes containing the 5'itr. similar data were obtained with probes containing the 3'itr. the rf of all the observed complexes was measured and plotted versus the position of the center of each probe. for each probe, two single end complexes were obtained, as expected. the sinus curves calculated from our data allowed us to map the center of the mos1-induced bend, located at the lowest point of the curve. this was at position for sec which located the bending core around the 21st nucleotide of both itr. for sec the lowest point of the curve was around position, which located the bending core around the 18th nucleotide for both itrs. these values are the means for at least five independent experiments. zhou's equation was used to determine bending angles of ° and ° for the sec formed with the 3' itr- and the 5' itr-containing fragments respectively, and ° and ° for the sec formed on the 3' itr- and the 5' itr-containing fragments respectively. in conclusion, our results indicated that both 3'- and 5'-itr were bent at angles of about 90° as a result of mos binding. interestingly, these angles may facilitate the assembly of a transposition complex in a minute transposon, such as the-one investigated here, since the stuffer fragment needs only form an angle of less than 90° to bring the transposon ends together which is quite possible for a 66-bp dna segment. another source of dna flexibility, such as single strand dna cleavage at the inner end of the itr, was also investigated. the cleavages produced by mos in sec and pec were therefore re-investigated, taking into account the inner part of the itr. we have already published that the itr is mainly cleaved at the 3'-end of the transferred strand and at positions + and + on the non-transferred strand, although several other cleaved positions were detected in the flanking dna on both strands. analysis of the bottom part of the gel also reveals that a single strand dna cleavage occurred on the transferred strand at position which is located at the inner end of the itr. impact of stuffer sequence on mos transposition two questions were addressed concerning the impact on transposition efficiency of the gc content of the transposon sequence, and of the number of ta dinucleotides in the transposon sequence. we first prepared nine pseudo-mos with gc contents ranging from to %. the reference construct was the pbc-3tet which has a gc content of %. in bacterial transposition assays, the transposition efficiency of the ten pseudo-transposons ranged over two orders of magnitude. the data were plotted, and the correlation between the two parameters was tested using the spearman correlation test with a significance threshold of. nine of the constructs plotted in this analysis were included in the ellipse, and the correlation between gc content and transposition frequency had a coefficient of about. this coefficient was significant enough to consider that there was a correlation between gc content and transposition frequency. however, the facts that one transposon construct fell outside the ellipse, and that two other transposon constructs with gc contents close to 56% differ by more than one order of magnitude indicates that gc content is not the only sequence parameter that affects the stuffer quality for transposition. we then prepared two modified genes encoding for tetracycline resistance: the first was ta-rich, and the second was ta-poor, but this did not have any marked effect on the percentage of gc. these two genes were used as markers in transposition assays in bacteria. it was expected that reducing the ta density would result in increased transposition efficiency, simply because some suicide auto-integrations would be avoided. we observed no correlation between the ta numbers, and the transposition frequency. this point was confirmed by the fact that a bleomycin resistance gene without the ta dinucleotide gave the same result in transposition assays in bacteria as the wild type bleomycin resistance gene. impact of temperature on mos transposition efficiency in vitro transposition assays were performed at four temperatures, and 37°c. our results are in agreement with those previously obtained for himar, as we found an optimal temperature of about 28°c, with no significant differences found between 28°c and 30°c. since we had previously shown that mos binds to its itr with similar efficiency regardless of temperature, we therefore checked whether a temperature effect was observed for mos excision or not. time course excision assays were performed either at or 37°c, using pbc-3tet plasmid as the transposon donor, and purified mbp-mos changing the excision temperature led to two main differences. the first was that, at 37°c, a marked dna degradation occurred, leading to the loss of about 50% of the dna two hours into the reaction, whereas no dna loss was observed after hours at 30°c. the second difference was that, despite the dna degradation, the pseudo-mos accumulated at 37°c, whereas it was not detectable at 30°c. this is not very surprising because an accurately excised transposon tends to be reinserted rather than accumulating. indeed, insertion products were seen in the assay performed at 30°c, but not in the assay performed at 37°c. this was in total agreement with the fact that more efficient transposition was observed at 30°c than at 37°c. the dna degradation and the 3tet accumulation detected at 37°c might be due to a single phenomenon: at 37°c, mos cleaves dna not only at the end of the itr but anywhere in the dna. this loss of specificity may promote the production of excised 3tet with extremities that are unable to integrate. organization of excised pseudo-mos produced at 37°c the organization of the pseudo-mos that accumulated at 37°c was analyzed using a variant of the pbc3tet pbc-3kana a transposon donor with a unique clai site in 3kana. in southern blot analyses, pbc-3kana is detected both as open circle and linear molecules, whereas the excised 3kana accumulates as a double-band of kbp. due to the sensibility of the technique, integration events are also detected. the detection of a double-band of about -kpb suggests that excision products could exist both in linear and circular configurations. southern analysis of the dna samples digested by clai was then monitored. once cleaved by clai, the band with the highest apparent molecular weight in undigested dna samples was shortened accordingly to the position of the restriction clai site. this suggests that this band corresponds to a linear excision product. in contrast, the band of lower intensity in undigested dna samples is unaffected or only slightly by clai digestion, thus suggesting that it corresponds to a circular excision product or that it had no clai site. primer name the excision products were therefore purified by agarose gel elution from untreated dna samples. they were submitted to pcr using primers anchored in an inverse orientation in the sequence of the kanamycin resistance gene, kanajunc and kanajunc, in order to amplify itr junctions looking like those isolated from bombyx mori cells transformed by mos1-based vectors. the amplification of a fragment of about 950-bp is expected if the circular forms correspond to the circularization of an excised full-length transposon. fragments ranging from to 800-bp were obtained and cloned. twelve were sequenced: they all contained the clai site, and a single itr, inserted into or close to the kanamycin resistance gene. this fact, combined with the fact that they were shorter than expected, indicates that these fragments resulted from intra-molecular integration events. among the twelve fragments analyzed here, four came from auto-integration events occurring in a dinucleotide that was not a ta but tt, tc, tg or ca dinucleotides. in conclusion, our data are consistent with the fact that some of mos excision products formed at 37°c are not able to perform integration into a target dna. this is due to the formation of linear products with inefficient extremities and circular products resulting from intra-molecular auto-integration. taken together, these events account for the decreased transposition efficiency observed at 37°c. impact of the transposon donor configuration on mos transposition in vitro transpositions were performed using a linear plasmid donor of transposon and compared to those obtained using a super coiled transposon donor. linearization of the transposon source dramatically reduced the transposition efficiency at 28°c, and hindered transposition at other temperatures. excision efficiencies of a super-helical donor were then compared to those obtained with the same donor linearized by ncoi. when the super-helical pbc-3tet was used as the substrate, products were digested by ncoi at the end of the experiment, making it possible to carry out direct comparisons. excision assays have been first analyzed by tracing the dead-end products of the reaction, i.e. bands α and δ. our data therefore show no major difference between the two reactions. analyses of the time taken to obtain 50% of α and δ fragments indicated that it was about and hours for the super-coiled and linear donors, respectively. furthermore, the curves differ in shape, the production of α + δ being faster with the circular substrate. this suggested differences between the two excision reactions. this difference was studied measuring the patterns of appearance of the β and γ fragments that are not dead-end products of the reaction. indeed, when mos excision occurs within the synaptic complex, β and γ are not expected to accumulate, because cleavage is believed to occur in a concerted fashion at both itrs. much more β and γ accumulated when the donor was linear, rising to a peak before decreasing, as the result of cleaving the second itr. in contrast, little β and γ accumulated when the substrate was super coiled, reaching a maximum of 10-fmoles. these data supported the idea that the concerted cleavage at both itrs is reduced or abolished when the substrate is linear, a phenomenon that may account for an overall decrease in the transposition efficiency. finally, transposition assays presented in fig. 5a corresponded to minutes reactions. at this time, excision and re-integration of the transposon from the circular substrate had already occurred whereas this had not occurred for the linear substrate. data in this section indicates that the excision is probably one of the limiting factors that accounts for the drastic decrease in transposition efficiency when the donor is linear. at least two non-mutually exclusive explanations can be proposed: excision from a linear donor is slower and excision from a linear donor takes place independently at both itrs. discussion in addition to requiring itr and the tpase, previous studies have demonstrated that the transposition of mle or tle requires critical parameters that interfere with transposition efficiency. these parameters include tpase concentrations, demonstrated in vitro for himar and mos and in vivo for mos, temperature, size of the stuffer, demonstrated in vitro for tc, sequence of the stuffer, demonstrated in vivo for mos, structural configuration of the target dna, demonstrated in vitro for tc. in this study, we have used an in vitro approach to address several parameters affecting mos transposition. parameters for optimal mariner transposition our results highlight the importance of the stuffer sequence since they show that the gc content is an important factor, even if it is probably not the only parameter that affects the stuffer performance for transposition. in silicio analyses failed to establish a correlation between dna curvature and/or flexibility and transposition efficiency. our results also confirm the impact of stuffer length on transposition efficiency, as already shown. length dependence of mos transposition might rely on auto-integration after excision. this is supported by the fact that large sophisticated transposons such as the phage mu or tn have specific mechanisms that prevent the transposon from inserting into itself. in contrast, simple elements such as mariners lack this function. a third parameter that controls mos transposition rate is the temperature. interestingly, elevated temperature promotes auto-integration events. auto-integration does not absolutely require a ta dinucleotide. this fact, associated with the fact that the percentage of ta dinucleotides does not influence mos transposition efficiency, rules out the possibility of designing large transgenes containing no ta to limit the length-dependence of mos transposition. mariner excision: making a molecule that produces efficient integration data presented here highlight an important, although not new, idea: transposition efficiency is in great part controlled by the quality of the excision product. this is in contradiction with published data that support the idea that cleavages at mles ends are far from accurate. however, all these data were collected in vitro, using linear, double-stranded oligonucleotides. on the other hand, mle in vivo excision footprints are often less variable, and closely match what the model predicts, i.e. hydrolysis at the 3' end of the element, and 5' break two or three nucleotides within the transposon. this suggests that experimental conditions can bias the description of excision product ends. our data allow proposing that there is a relationship between the super-helicity of the donor dna, and the production of efficient excision products, through the assembly of the synaptic complexes. indeed, mos excision from a super-coiled donor yields transposition that is 1000-fold more efficient than that resulting from a linear donor plasmid. these findings are fully consistent with super-helicity acting as a regulatory factor of mariner transposition in vitro, and probably in vivo. from this standpoint, mariner looks like many other transposons. for instance, mu transposition requires dna super coiling in the donor dna whereas tn needs either dna super coiling in the dna substrate, or ihf acting as a "super coiling relief factor". super coiling is probably not required for the chemical steps of the transposition, but might be a limiting factor for the assembly of mariner transposition complexes. although mariner transposition has been shown to take place in vitro, we cannot rule out the possibility that host factors may assist the transposase during in vivo transposition. mite transposition mites are small, repetitive, dna elements ranging in size from approximately to bp that are interspersed within eukaryotic genomes. their sequence is palindromic, at-rich, and has no coding capacity. for their mobility, they generally use a source of tpase in trans, originating from a related complete transposon copy. our data indicate that the size of mites of mle origin does not limit their ability to transpose. for the smallest ones, found in the human genome or in that of the ant messor bouvieri, the binding of mos causes major itrs bending, thus bringing the two ends of the transposon close to each other. in addition, the inner itr cleavage by mos could also allows the itr bending. similar cleavage was found on the non-transferred strand of the hsmar itr cleaved by hsmar no host factor is therefore required to bend the dna and assemble the synaptic complex. this model provides a very satisfying explanation of the mobility of the mle mites, because it explains how a minute mos can assemble a functional synaptic complex similar to that of the full-length mle. moreover, it is supported by in vitro transposition data. however, it does not explain why most of the mites, including those derived from mle, display a palindromic organization throughout their sequence. in contrast to what was previously proposed, our data suggest that complete palindromy is not a requirement for mite mobility, and that it could therefore result from other constraints.  non-viral vectors are presently invading the world of gene transfer and mutagenesis. among the tools that are currently under evaluation, the transposon mos raises a growing interest. in fact, it is perceived as a small versatile and quasi-universal means of transforming a variety of cells, except mammal ones. however, as for any new tool, it is necessary to control every parameter driving its transposition, and, in a first place, the factors that might limit its efficiency and specificity for dna transfer. in the present work, we have investigated several parameters, particularly those related to the structure of the transposon. in this respect, we have demonstrated that the dna sequence as well as the length of the inner part of the transposon have a crucial effect on the transposition efficiency. our results provide, for the first time, an experimental in vitro demonstration that mites of mle origin that are as small as 80– bp are able to transpose. hence, we propose a model that bypasses the physical constraints associated to the transposition of such small transposons. we have also demonstrated that the super-helicity of the transposon impacts considerably on the efficiency of transposition. finally, we have found that mariner transposition is dependent on the temperature. the fact that higher temperature disfavors re-integration of the excision products might be related to the transposase enzymatic properties. 
we provide the first in vitro experimental demonstration that mites of mle origin, as small as to 120-bp, are able to transpose. finally, the super-helicity of the dna transposon donor has a dramatic impact on the transposition efficiency. in vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the mles transposition in vivo. the study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. mariner-like elements are widespread dna transposons in animal genomes. excessive temperature down-regulates mos transposition, yielding excision products unable to re-integrate. the transposition of mos first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the dna stuffer, and its length.
 mariner-like elements are widespread dna transposons in animal genomes. although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the mles transposition in vivo.  the transposition of mos first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the dna stuffer, and its length. we provide the first in vitro experimental demonstration that mites of mle origin, as small as to 120-bp, are able to transpose. excessive temperature down-regulates mos transposition, yielding excision products unable to re-integrate. finally, the super-helicity of the dna transposon donor has a dramatic impact on the transposition efficiency.  the study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. in vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. we propose a model for small transposons transposition that bypasses dna bending constraints. 