transposons employ intricate mechanisms of excision and reinsertion , and they appear to play an important role in the evolution of their hosts , often accounting for a substantial part of the genomes [ 1 - 5 ] .
more recently , however , they have primarily attracted attention as tools for the manipulation of the genomes of their natural hosts and , more importantly , the genomes of other species that are not closely related .
transposons have even been found to be active in organisms that do not belong to the kingdom of their natural host .
this review summarizes the advances that have been made with one of these versatile genomic tools , namely the transposon minos .
minos is a dna ( class ii ) transposon that was fortuitously identified as a repetitive element in the genome of the fruit fly drosophila hydei .
the natural element , approximately 1.8 kilobases in length , is characterized by 254 base pair ( bp ) perfect inverted terminal repeats flanking a two - exon transposase gene ( figure 1 ) .
the minos element carries a single gene that is interrupted by a 60 bp long intron and encodes a transposase .
the amino - terminus of the transposase contains a putative dna - binding domain resembling the paired domain , a conserved feature of the pax protein family .
the carboxyl - terminus of the minos transposase contains a d , d34e catalytic triad , which is also found in transposases of related elements such as tc1 and bari1 .
the presence of an unmapped nuclear localization signal has been inferred based on the nuclear localization of a minos transposase - enhanced green fluorescent protein ( egfp ) fusion .
idr , inner direct repeat ; itr , inverted terminal repeat ; odr , outer direct repeat .
surveys among drosophila spp . have revealed widespread occurrence of minos - like elements ; 21 out of 26 analyzed species of the repleta group and 5 out of 7 analyzed species of the saltans group carry minos - like transposons .
evolutionary analysis suggests that the distribution of minos in the genus drosophila is best explained by horizontal transfer of the element across species .
like most dna transposons , minos moves in a host genome with a cut - and - paste mechanism , whereby the transposase excises the element from the original site of insertion and reinserts it into a new locus in a nonreplicative manner .
the transposition of minos , like that of the other tc1/mariner - like elements , occurs into a ta dinucleotide that is duplicated upon insertion ; this implies that a staggered cut of the target dna leads to 2 bp single strand ta overhangs as the first step in the insertion reaction .
analysis of the sequences that flank insertion sites in the genome of drosophila melanogaster revealed that minos transposase has little sequence preference beyond the ta target dinucleotide .
this is in contrast to most other transposable elements studied thus far , which exhibit variable degrees of flanking sequence preference , resulting in biased insertion and consequently the existence of ' hot ' and ' cold ' spots along the genome .
although much of the recent work on transposons is concerned with the transgenesis of vertebrates , simpler model organisms and cell lines are more suitable for the analysis of the transposition mechanism .
the introduction of a non - autonomous minos element and of a transgene expressing transposase into the d. melanogaster genome   allowed the study of the minos transposition mechanism by mobilization of the non - autonomous element and subsequent molecular analysis of the excision and transposition events .
two types of chromosomal sites were recovered after excision : sites precisely restored and sites containing leftovers ( footprints ) of the mobilized element . precise excision ( restoration of the original site of insertion ) was detected only in flies heterozygous for the insertion , presumably resulting from gene conversion .
most footprints were 6 bp long and consisted of the four terminal nucleotides of either one of the inverted repeats plus the duplicated ta dinucleotide .
the structure of the footprints suggested a possible mechanism of excision involving staggered cuts by the transposase at the ends of the inverted repeats , creating single strand overhangs of the four terminal bases of each end . during reinsertion
, the overhangs of the mobilized element are joined to the ta overhangs of the target site , followed by polymerase - mediated fill - in of the single stranded dna .
this model predicts that a heteroduplex flanked by the ta duplication is formed at the site of excision .
indeed , the existence of such heteroduplexes in the genome of d. melanogaster flies in which minos is actively excising was directly demonstrated .
several excision sites were also cloned from vertebrate cells , in which a single non - autonomous minos element was mobilized by trans acting transposase .
these footprints , however , were much more heterogeneous in their size and structure , reflecting differences in the post - excision processes compared with drosophila .
an analysis of minos excisions from a plasmid in the simple chordate ciona intestinalis identified the typical 6 bp footprints as well as variants thereof with short insertions or deletions .
sequence - specific interaction of transposase and the transposable element at or near the inverted repeats is required for transposition .
each of the minos inverted repeats contains two 18 bp direct repeats near the ends of the inverted repeat ( figure 1 ) .
dnaasei protection with purified recombinant minos transposase protein shows that these direct repeats are bound by transposase ( figure 2a ) .
binding of transposase to the minos inverted terminal repeat and internal minos transcription initiation sites .
g+a reaction ' indicates g and a specific sequence reaction ; the dnaasei protection reactions were done in duplicate . ' + transposase ' indicates twofold molar excess of transposase over target dna , and ' + + transposase ' indicates 20-fold molar excess of transposase .
the positions of the protected sequences ( open boxes ) , the hypersensitive positions ( filled box ) , as well as the positions of the inner ( bottom arrow ) and outer direct repeats ( top arrow ) are indicated .
a ' , ' c ' , ' g ' , and ' t ' indicate base specific sequencing reactions . also shown
are primer extension reactions with a minos - specific primer on total rna isolated from d. melanogaster populations with ( + ) or without ( - ) a minos insertion .
the lower horizontal arrow indicates a transcription start about 30 base pairs downstream from the inner direct repeat .
an additional transcription start site is detected between the two direct repeats ( upper horizontal arrow ) .
although the binding site close to the end of the element ( the ' outer ' direct repeat ) is expected to play a role in the excision of the element , the function of the second , ' inner ' repeat is not known .
in addition to minos , two other well studied transposons of the tc1/mariner family carry two direct repeats per inverted terminal repeat : tc3 and sleeping beauty .
transposon mobility assays suggest that both the outer and the inner pairs of binding sites are required for full activity of sleeping beauty   and minos ( klinakis a and savakis c , unpublished data ) but not that of tc3 .
the mechanism underlying the involvement of the inner direct repeat in minos and sleeping beauty transposition remains to be elucidated .
it is intriguing that the inner direct repeat overlaps a putative tata box in the 5 ' inverted repeat of minos , and primer extension analysis revealed a transcriptional initiation site for the minos transposase approximately 30 bp downstream of this repeat ( figure 2b ) .
these observations suggest a possible role for the inner transposase binding site in autoregulation of transposase expression .
negative feedback mechanisms that regulate transposition have been suggested for other transposable elements too [ 23 - 25 ] . in vitro activity assays established for other members of the tc1/mariner transposon superfamily
although direct biochemical evidence is not yet available for minos , the ability of minos - based vectors to transpose in cells of a wide range of metazoan species [ 19,28 - 40 ] is indicative of the element 's host independence ( figure 3 ) .
assays for three types of minos activity are distinguished and indicated ( extrachromosomal , chromosomal , and germline ) .
transposon mediated transgenesis mimics nature ; many transposons found in genomes are non - autonomous derivatives .
non - autonomous elements also carry functional inverted repeats but no functional transposase gene , because of the accumulation of point mutations and deletions .
elements that lack an intact transposase gene are unable to catalyze their own transposition , but they can be mobilized with transposase supplied in trans . in a typical engineered transposon , the transposase gene is inactivated through the insertion of a selective marker or deleted and replaced by a marker gene .
transposase is supplied in trans from a copy of the transposase gene introduced into the genome , or ( more often ) expressed from co - injected plasmid or transposase mrna .
the transient nature of the supply of transposase is the key to the generation of stable insertions .
a construct carrying a non - autonomous transposon is usually termed a ' donor ' and a construct carrying an active transposase gene is termed a ' helper ' . the minos transgenesis system is supported by an extensive toolkit that has allowed experimentation in a wide spectrum of species .
first , extra - chromosomal assays have been devised to facilitate and speed up the process of assessing the effectiveness of the minos components in diverse organisms of interest .
second , an ever - increasing number of minos - based transformation vectors has been created , containing either fluorescent proteins such as gfp or more traditional marker genes for the easy identification of integration events and transgenic individuals [ 14,17,30 - 32,35 - 37,39,40,42 - 44 ] .
third , the availability of different transposase sources , varying from a chromosomal copy of the transposase gene to a helper plasmid or in vitro synthesized mrna , can fit most purposes and experimental settings .
the development of minos - based gene manipulation for mammals was based on a first phase of evaluation in insects . as in the case of all other insect transposons
, d. melanogaster has been the favored testing ground for most components and applications of the minos system .
the original use of minos as a germline transformation vector in d. melanogaster   was followed by the transformation of the medfly ceratitis capitata , in the first report of a transposon system applied in a genus other than the one in which it was originally identified .
the validation of improved minos tools in activity assays conducted in mosquito and lepidopteran embryos and cell lines   opened the way to the stable germline transformation of a species of great medical importance , namely the human malaria vector anopheles stephensi .
more recently , the development of even more efficient minos transposase sources   and of versatile fluorescent marker systems   facilitated the genetic transformation of diverse arthropod species , ranging from diptera ( the olive fly bactrocera oleae ) to coleoptera ( the red flour beetle tribolium castaneum ) , lepidoptera ( the silkworm bombyx mori ) , and crustacea ( the amphipod parhyale hawaiensis ) .
the genetic transformation of d. melanogaster using the p - element over 25 years ago   triggered the development of innovative experimental approaches that revolutionized molecular genetic analysis in this model organism .
however , the use of the p - element is limited to species closely related to d. melanogaster .
the establishment of minos and other transposon systems allowed a number of these forward and reverse genetic methodologies to be transferred to several non - drosophilid insects of experimental and practical interest , as well as to a crustacean .
for instance , analysis of cis regulatory elements with reporter constructs has been demonstrated in ceratitis and parhyale , enhancer trapping has been undertaken in tribolium   and parhyale ( pavlopoulos a , averof m , unpublished data ) , conditional mis - expression systems have been developed in anopheles and parhyale   ( pavlopoulos a , averof m , unpublished data ) , and post - transcriptional gene silencing via rna interference constructs has been employed in anopheles .
minos activity is not limited to arthropod species ; it has also been used for germline transformation and enhancer trapping in two species of ascidians .
finally , minos - based transposons have been mobilized successfully in cultured human cells and in the mouse soma and germline .
in addition to minos , there is a handful of other dna transposons used for vertebrate transgenesis , the most widely employed of which is sleeping beauty .
the relative evaluation of these different transposons is difficult unless experiments are performed under comparable conditions , which is generally not the case .
however , one such comparison has been done with regard to transposition efficiency of the transposons sleeping beauty , tol2 , mos1 , and piggybac . in all four different vertebrate cell lines
one should , however , be careful not to take the transposition rate as the only criterion of the usefulness of a transposable element as a genomic tool .
depending on the intended application , insertional preference , the capacity to carry large inserts , mutagenicity , the ability to create deletions upon excision , and other properties can be at least equally important as high transposition frequency .
the availability of sequenced genomes for several model and nonmodel organisms signals the next challenge in genomics research , namely the functional annotation of all predicted genes .
classic genetic analysis relies on the use of mutations to study the function of the affected gene .
the most common experimental approach is inactivation of the gene by chemical mutagenesis or insertional mutagenesis using transposable elements or viruses .
insertional mutagenesis has the advantage that the targeted gene is at the same time tagged with the inserted dna , and so it is relatively easy to identify and retrieve . in principle , the short target sites of most transposable elements would allow a single transposon to saturate the whole genome with mutagenic insertions . in practice , however , every transposable element exhibits insertional biases for certain genomic loci , rendering mutagenesis of the entire genome problematic .
for example , the ability of the p - element to target new genes in d. melanogaster appears to have reached its limits . in accordance with the distinct target preferences between minos and p
, a pilot study   indicated that more than half of minos generated insertions were in genes not previously hit by the p - element .
although the p - element tends to insert in 5 ' untranslated regions , minos insertions in drosophila genes occur preferentially into introns .
these insertions appear to be spliced out with the targeted introns and thus do not interfere with normal gene function .
however , re - mobilizing such insertions by providing transposase in trans leads at reasonable frequency to deletions of flanking dna , so that induced excision can be used to ' knockout ' the targeted gene .
because of the ability of minos to transpose at high frequencies and the fact that 60% of all minos insertions were in or close to genes , the minos system has become an integral component of the drosophila gene disruption project , which is a systematic effort to mutate all genes in the drosophila genome   ( bellen h , personal communication ) .
the efficiency of large - scale mutagenesis screens is enhanced in genetic schemes that utilize two different transposons , say a and b. the ' jump starter ' element a ( with inverted repeats a ) encodes transposase b that , when expressed , mobilizes the ' mutator ' element b ( with inverted repeats b ) into new genomic loci .
the independent development of both minos and piggybac based gene transfer systems in the beetle tribolium castaneum   allowed the initiation of a large - scale insertional mutagenesis screen by the geku consortium ( research groups based in goettingen , erlangen , kansas , and the us department of agriculture grain marketing and production research center ) .
in such screens , minos might be more useful as the mutator element because , unlike piggybac , it can excise imprecisely , offering the possibility to generate mutant alleles secondarily .
other transposon based approaches to genomic analysis include the various ' trapping ' schemes , in which expression of a reporter or selection gene carried by the inserted transposon depends on the targeted gene . in enhancer trapping   the expression of a transposon encoded marker gene is activated or modulated by transcription enhancer elements near to the transposon insertion site . in such traps ,
expression of the marker gene follows partly or fully the spatial and temporal expression of the gene normally controlled by the ' trapped ' enhancer elements .
minos based enhancer trapping has been demonstrated in the ascidian ciona , in tribolium , in parhyale ( pavlopoulos a , averof m , unpublished ) , and in d. melanogaster ( metaxakis t , savakis c , unpublished data ) . in exon
trapping , expression of a transposon - encoded marker gene depends on appropriate splicing of the marker gene upon insertion of the element into an intron .
the preference of minos to insert into introns in the fly and the mouse   ( zagoraiou l , unpublished data ) suggests that minos may be particularly useful for this type of analysis .
indeed , the choice of an exon trapping minos based transposon as mutagen in human cells in culture was justified by the outcome of the experiment .
this transposon carried an intronic splice acceptor followed by a selection gene that would be expressed only upon insertion into an intron of an active gene and then spliced in the correct frame to the upstream exon . using this
transposon mediated mutagenesis ( tramm ) method , it was demonstrated that the minos system can potentially trap / tag all genes in a mammalian genome .
co - transfection of a minos transposon construct with a helper plasmid expressing transposase into hela cells resulted in 4% of the cells being stably transformed , with on average two minos insertions per transformed cell . in later studies , efficient inter - chromosomal transposition of minos
was demonstrated in double transgenic mice , carrying tandem copies of non - autonomous minos elements and the minos transposase gene under the control of tissue specific promoters .
the observed transposition rate was much greater in oocytes than in lymphocytes ( 8% versus 0.6% ) .
its high germline activity makes minos a powerful tool for genome - wide functional studies in whole mice and probably in other vertebrates .
minos has attracted interest as a tool for basic research , for biotechnological purposes , and for developing strategies to control arthropod disease vectors and agricultural pests [ 30 - 32,51 ] .
minos has been successfully evaluated as a tool for the transgenesis of diverse animal species , both vertebrates and invertebrates , whereas its functionality in plants has yet to be tested . in most species
tested , minos transposition occurs with high frequency , transposons can carry relatively large transgenes , insertions are stable in the absence of transposase , and the only obvious target site preference is the ta dinucleotide .
overall , these features demonstrate the usefulness of minos as a tool for functional genetic and genomic approaches in model and nonmodel organisms , including vertebrates .
cs is a founding member and nonexecutive director of a company , minos biosystems ltd , which owns intellectual property related to the minos transposable element .
this article has been published as part of genome biology volume 8 , supplement 1 , 2007 : transposons in vertebrate functional genomics .
 transposons are powerful tools for conducting genetic manipulation and functional studies in organisms that are of scientific , economic , or medical interest 
 . minos , a member of the tc1/mariner family of dna transposons , exhibits a low insertional bias and transposes with high frequency in vertebrates and invertebrates . 
 its use as a tool for transgenesis and genome analysis of rather different animal species is described . 
Introduction
History of 
The mechanism of transposition
None
None
Conclusion
Competing interests
Acknowledgements