BACKGROUND
the thermodimorphic fungus paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic endemic disease that affects at least  <dig> million people in latin america  <cit> .
infection probably occurs as a result of inhalation of conidia that subsequently transform into yeast forms within the lung, resulting in asymptomatic infection or infection that can progress to acute  and chronic clinical forms of the disease  <cit> .
paracoccidioidomycosis is the 8th most common cause of death due to chronic/recurrent infections and parasitic diseases in brazil  <cit> .
the teleomorph  stage of this fungus is unknown, but analysis of the large rdna subunit classified it as an ascomycete, order onygenales, family onygenaceae, phylogenetically close to ajellomyces, a genus that harbors  <dig> other human dimorphic fungal pathogens, specifically histoplasma capsulatum and blastomyces dermatitidis  <cit> .
molecular analysis by random amplification of polymorphic dna   <cit> , restriction fragment length polymorphism   <cit>  and electrophoretic karyotyping showed extensive genetic variability among distinct isolates  <cit> .
rapd, a technique popular for its simplicity, has been useful in determining the existence of genetically distinct p. brasiliensis groups, their relationship with geographic distribution, and the similarity among clinical, animal and environmental samples.
however, the information has been difficult to compare.
gene polymorphism studies were pioneered by characterization of pbgp <dig> polymorphism, a gene encoding the fungus immunodominant antigen, in a sample of  <dig> isolates  <cit> .
by comparing  <dig> sequenced pcr fragments from the whole gene  these authors found  <dig> informative substitution sites, mostly in exon  <dig>  which defined 5- <dig> genotypes.
the maximum-likelihood phylogenetic tree generated with these sequences clearly reflected the presence of specific genetic groups in the species.
subsequently, multilocus genealogy studies demonstrated evolutionary lineages identifying the occurrence of cryptic phylogenetic species that were morphologically indistinguishable by following the criteria of genealogical concordance and non-discordance  <cit>  which is used to identify the reduction of gene flow between groups of individuals due to geographic or reproductive isolation barriers.
the pbgp <dig> locus, whose characteristic genotypes have recently been reviewed  <cit> , was the most polymorphic and informative in these studies.
matute et al.
<cit>  analyzed the genetic structure of  <dig> p. brasiliensis isolates and concluded that they could be grouped into  <dig> distinct phylogenetic species: s <dig> , ps <dig>  and ps <dig> .
in a study of  <dig> p. brasiliensis isolates,  <dig> of which had been included in the above study, carrero et al.
<cit>  came to a similar conclusion for all the isolates, with the exception of isolate pb <dig>  which they suggested was a new phylogenetic species in the genus paracoccidioides.
recently, teixeira et al.
<cit> , analyzing  <dig> isolates of the fungus, found that  <dig> of them were genotypically similar, belonging to the pb01-like group.
they estimated that the s1/ps2/ps <dig> species clade and the pb01-like new species, for which the name paracoccidioides lutzii was proposed, shared a common ancestor approximately  <dig> million years ago.
these studies suggested that the genus paracoccidioides consists of at least  <dig> different, previously unrecognized phylogenetic species: p. brasiliensis s <dig> , ps <dig>  and ps <dig> , and a 4th quite distinct lineage comprising fungal isolates formerly referred to as pb01-like  <cit> , now proposed to be a new species, p. lutzii  <cit> .
the multilocus sequence analysis also supported recombination in nature in p. brasiliensis   <cit>  and p. lutzii  <cit> , indicating the presence of sexual reproduction.
thanks to the broad institute fungal genome initiative  project, the complete genome of representative isolates from p. brasiliensis s <dig> , ps <dig>  and pb <dig> like  have recently been made available at the site: http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis thus allowing further and specific comparisons among isolates.
previous local projects, however, contributed to the construction of large expressed sequence tag databases of pb <dig>  <cit>  and pb <dig>  <cit> .
transposable elements  represent a substantial fraction of eukaryotic genomes that have been found in virtually all species investigated to date.
they are abundant in fungi and represent 3-20% of the genomes of many filamentous fungi  <cit> .
tes can produce a variety of effects in genomes, such as gene inactivation or modification, chromosome breakage, and genome recombination or rearrangement, thereby generating plasticity  <cit> .
karyotyping and gene mapping indicated the occurrence of chromosomal rearrangements  <cit> , which are associated with the presence of repetition and te in fungal genomes  <cit> .
tes can be grouped into  <dig> classes according to their transposition mechanism.
class i consists of retrotransposons that transpose via an rna intermediate that is reverse-transcribed into dna by a te-encoded reverse transcriptase.
class ii elements, also called dna transposons, move via a dna intermediate.
because of the abundance and diversity of tes described in the eukaryotic genomes, a unified classification system for eukaryotic tes has been proposed by different groups  <cit> .
dna transposons can be divided into  <dig> subclasses: the first comprises the elements that transpose via the classical "cut-and-paste" mechanism; the second comprises elements that transpose by replication involving the displacement of only one strand; and in the third, transposons move by a mechanism called "rolling-circle"  <cit> .
subclass  <dig> dna transposons require the cleavage of both dna strands for the transposition and have  <dig> orders - tir and crypton.
six superfamilies of the order tir from subclass  <dig> have been identified in fungi: tc1/mariner, hat, mutator, transib, pif-harbinger and cacta.
tir transposases contain a signature active site motif that consists of  <dig> acidic amino-acids  and forms the catalytic pocket of the enzyme responsible for cleaving dna strands.
crypton, the only superfamily representative of the second order, has only been found to date in fungi and, unlike any previously described transposons, encodes a putative tyrosine recombinase instead of a classical transposase.
this recombinase lacks an rt domain, which suggests that transposition occurs via a dna intermediate.
it does not contain long repeat sequences, but is bordered by short direct repeats that may have been generated by its insertion into the host genome by recombination.
cryptons may be excised from the host genome by tyrosine recombinase, generating a circular, extrachromosomal, double-stranded dna molecule, for which cleavage of both dna strands is required.
elements from subclasses  <dig>  and  <dig>  have only one strand cleaved during transposition and have also been identified in fungi, but the tc1/mariner superfamily is probably the most prevalent in these organisms  <cit> .
transposons contain terminal inverted repeats , which are recognized by the pipsqueak helix-turn-helix  motif in the transposase domain.
each subclass  <dig> dna transposon superfamily is characterized by a specific transposase core that is different from those of other superfamilies.
upon insertion, dna transposons generate target site duplications  with lengths that are relatively well conserved among superfamily members  <cit> .
tes have been poorly characterized in p. brasiliensis.
transcriptome analysis indicated that ty-like retroelements were among the most abundant ests in p. brasiliensis  <cit> .
nascimento et al.
<cit> , searching for microsatellites that could be potentially useful as molecular markers, reported that a small number of them were located in transposons.
the focus of our work has been the characterization of dna transposons and their derivatives in the genus paracoccidioides.
to this end we searched for dna transposons in the paracoccidioides genomic sequence database http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis.
we have discovered  <dig> new families of dna transposons and one new subfamily of mariner-1_af belonging to the tc1/mariner superfamily.
remarkably, some trem  elements appear that may be active in the genomes of representative isolates of  <dig> different paracoccidioides phylogenetic lineages.
the finding of active autonomous dna transposons in p. brasiliensis may have implications for an understanding of the evolutionary processes underlying the diversification of this group.
furthermore, transposons are efficient vectors for introducing foreign dna into cells.
RESULTS
occurrence of dna transposons in the genus paracoccidioides
a survey of the p. brasiliensis pb <dig> functional and differential genome project annotated est database   <cit>  for sequences similar to transposase sequences found  <dig> contigs  that contain open reading frames  encoding proteins of  <dig> and  <dig> amino acids, respectively, with similarity to the conserved hth dna binding domain of the transposase of the aft <dig> dna transposon in aspergillus fumigatus.
these sequences were amplified from p. brasiliensis genomic dna, their identities confirmed by sequence analysis, and then used as probes in southern blot hybridization with genomic dna of different p. brasiliensis isolates.
this revealed the presence of multiple copies of dna transposons dispersed throughout the genome of different isolates.
rt-pcr analysis of total rna of the isolates pb <dig>   <cit> , pb <dig> and pb <dig>   <cit>  confirmed transcription of the putative elements .
the transposon sequence from each est contig was used as a query in a modified blastn search  against the whole genome sequence assemblies of all  <dig> paracoccidioides genomes  and combined with the strategy for identification of tirs, what allowed the artemis annotation tool to be used to identify transposons.
adna transposase sequences from the p. brasiliensis transcriptome were used to perform queries in a modified blastn search  in the genome of isolates pb <dig>  pb <dig> and pb <dig> and combined with a strategy for identification of tirs.
belements assigned to a dna transposon superfamily according to the standard principles of the te classification proposed by wicker et al.
<cit> .
csequences that do not meet all the criteria proposed by wicker et al.
<cit> .
they are highly defective elements, with lower conservation of the typical transposase domains .
we also searched the paracoccidioides genomes for cryptons, rolling-circle dna transposons  and self-synthesizing dna transposons   <cit> , using consensus sequences for these elements , no significant matches were found.
in the genomic sequence of pb <dig> , only  <dig> dna transposon families have been classified, corresponding to  <dig>  of the  <dig> hits found in this isolate .
in the set of elements for pb <dig>   <dig> full-length potentially functional autonomous elements coding for putative transposases with well-defined tirs were classified.
the copies of the  <dig> elements found in pb <dig> totaled  <dig> hits, or  <dig> % of the  <dig> hits, similar to dna transposons in this isolate .
of the  <dig> hits registered in pb <dig>   <dig>  were identified and correlated with tc1/mariner elements.
tc1/mariner superfamily in the genus paracoccidioides
we identified  <dig> dna transposon elements in paracoccidioides that share 65-80% identity at the nucleotide level with tc1/mariner transposons deposited in repbase  <cit>   at the genetic information research institute .
like the tc1/mariner transposons,  <dig> of these elements are flanked by ta dinucleotide .
because of their similarity to the tc1/mariner superfamily and the fact that most of them  insert specifically in ta target sites, these elements were given the name trem .
the completeness of transposons was confirmed by the presence of tirs at both ends, followed by multiple sequence alignment to well-studied examples .
incomplete sequences were not included in the comparative analyses shown in figure  <dig>  we examined  <dig> insertion loci of trem elements , of which  <dig>  are flanked by well-defined tirs, while the remaining sequences, including all  <dig> tremg elements, are not flanked by tirs.
atsd - target site duplication.
ta dinucleotide.
brepeat masking - results of the analysis performed with the repeat masking script using the girinst http://www.girinst.org/repbase database and percentage similarity.
csize - total length of element in base pairs
dprotein - length of transposase in amino acids .
some elements contain  <dig> or  <dig> orfs encoding different proteins .
etirs - length of terminal inverted repeats in base pairs .
fcopy number - number of te copies found in the genome of the  <dig> p. brasiliensis isolates.
the number of elements with potentially functional orfs is shown in parentheses.
gn - the total number of tc1/mariner transposons identified in p. brasiliensis.
there are  <dig> copies of trema in the pb <dig> isolate,  <dig> which contain a full-length orf and  <dig> which are truncated, and  <dig> copies in the pb <dig> isolate,  <dig> which have an intact orf .
trema sequences were aligned to build an  <dig> bp consensus containing a single orf  that encodes a  <dig> amino acid protein with  <dig> conserved domains characteristic of transposases - hth, cenpb and dde .
the trema tirs are  <dig> bp long and have a  <dig> bp internal direct repeat .
analysis of trema with the repeatmasker script in the girinst database http://www.girinst.org/repbase revealed a similarity of only 66% with the a. fumigatus mariner-1_af transposon, suggesting that it may be a new dna transposon family  <cit> .
we found  <dig> copies of tremb,  <dig> in the genome of the pb <dig> isolate and  <dig> in pb <dig> .
however, only  <dig> copies showed intact orfs, which were used to generate a  <dig> bp consensus sequence flanked by  <dig> bp tirs .
this orf  encodes a  <dig> aa transposase with  <dig> conserved domains .
analysis using repeatmasker gave an index of 65% similarity with the mariner-1_af.
it is interesting that  <dig> tremb sequences carrying truncated orfs are flanked by intact tirs.
tremc is the most abundant dna transposon, having  <dig> truncated copies dispersed throughout the genomes.
the  <dig> longest sequences were aligned to generate a  <dig> bp consensus flanked by  <dig> bp tirs .
it contains  <dig> short orfs,  <dig> which encode the hth and dde domains .
the central region of the element shows similarity with a. fumigatus transposases, and analysis by repeatmasker revealed 66% similarity with mariner-2_ao in aspergillus oryzae.
the tremc shown in figure  <dig> is based on the element's most conserved sequences.
tremd is present in a small number of copies , exclusively in the pb <dig> isolate .
the consensus sequence has  <dig> bp and harbors a  <dig> bp orf  flanked by  <dig> bp tirs .
the orf codes for a  <dig> aa transposase carrying the hth and dde domains .
repeatmasker analysis showed 68% similarity with mariner-1_af in the central region, and 80% similarity with the tirs, suggesting that tremd is related to or belongs to this family  <cit> .
the consensus sequence for the treme transposon, found exclusively in the pb <dig> isolate, is  <dig> bp long, flanked by  <dig> bp tirs .
it includes  <dig> orfs: one has  <dig> bp  and codes for a  <dig> aa transposase with the hth and dde domains; and the second  encodes a  <dig> aa protein similar to a hypothetical protein  from ajellomyces capsulatum .
treme showed a 65% similarity with the mariner-1_af transposon.
with its  <dig> bp consensus sequence, tremf is the longest of the elements described in this study and the third most abundant  .
the consensus sequence is flanked by  <dig> bp tirs and contains  <dig> orfs: the first is  <dig> bp long  and codes for a  <dig> aa protein harboring the conserved domain cenpb; the second extends from position nt  <dig> to  <dig>  and codes for a  <dig> aa dde-domain-harboring protein .
the results of repeatmasker analysis revealed a 67% similarity with mariner-9_an in emericella nidulans, which is a non-autonomous dna transposon belonging to the pogo clade.
thus, tremf could represent a new transposon family  <cit> .
tremg, the 7th tc1/mariner element we identified, has  <dig> copies in pb <dig> and  <dig> in pb <dig> .
all copies are truncated, flanked by highly divergent and irregular sequences .
the central core of some elements has an orf coding for a  <dig> aa protein carrying the dde domain.
unfortunately, it was not possible to determine the precise boundary of this element because of the divergence among the flanking sequences.
repeatmasker analysis showed a 66% similarity with the mariner-5_af in a .fumigatus, a dna transposon of the tc <dig> clade.
for this reason, this element was classified as a tc1/mariner transposon.
tremh is present in the  <dig> isolates, with  <dig> copies in pb <dig>   <dig> in pb <dig> and  <dig> in pb <dig> .
of the  <dig> copies of this element,  <dig>  are flanked by  <dig> bp tirs; however, it was not possible to define its target site.
we used  <dig> copies that had the best sequence conservation  and encoded the dde motif to rebuild the  <dig> bp consensus sequence .
repeatmasker analysis showed a 66% similarity with the mariner3_ao in a .oryzae.
the element copy contains an orf coding for a  <dig> aa protein carrying the dde motif , similar to that in the transposon in talaromyces stipitatus  .
in this region,  <dig> strictly conserved acidic amino acids - d, d and d  - were found, and these probably constitute the dde catalytic motif in tremh.
since the transposase activity in many dna transposons relies upon the catalytic activity of the dde domain, we suggest that tremh is a tc1/mariner-like transposon.
phylogenetic analysis
the alignment of the predicted sequences corresponding to each of the conserved transposase domains found in  <dig> dna transposons in paracoccidioides, with the sequences of well-characterized dna transposons of the tc1/mariner superfamily  from other species, allows the identification of motifs considered essential for active transposition .
some of the paracoccidioides transposases are interrupted by stop codons, minor indels or the absence of a translation initiation codon .
the complete catalytic triad dde occurred in  <dig> of the elements  and was characterized by the substitution of the glutamate residue by aspartate to form a d, d, d motif, as previously observed in other fungal transposases.
the triad signatures  are very similar and can be represented by d10--d122--d <dig> in the elements trema, b, d and e; d10--d120--d <dig> in the element tremg; and d7--d119--d <dig> in the element tremh.
in tremf, the first aspartic residue was deleted, but the distance between the second and third aspartate residues was maintained -d125).
in tremc, all  <dig> aspartic residues were absent.
in addition to the conserved catalytic triad ddd, transposases contain a number of other highly conserved, group-specific amino acids, such as the hth and cenpb domains.
the hth domain was found in  <dig> trem elements  with varying degrees of conservation - 58% identity between the pairs trema/tremb, tremb/tremc and tremd/treme, and 53% identity between trema/tremc.
the identities between treme/trema and treme/tremb were lower than 40%.
alignment of the dna-binding domains of the transposases revealed a distinction between trem elements, which may be functionally connected to the tirs of these elements.
when the hth domain was compared with that of other fungal transposases, the highest identity index  was found between tremd and the tan <dig> transposon of aspergillus niger.
the cenpb domain was present in  <dig> of the paracoccidioides transposons .
the identity between trema and tremb was  <dig> %, and between these elements and trem f less than 36%.
it was also observed that  <dig> elements  presented conserved cx2cx4hx4c zinc finger domains in the c-terminal ends of their transposases corresponding, respectively, to amino acid residues 531- <dig>  528- <dig> and 540- <dig>  in tremc and treme, similar but non-canonical motifs were present at the c-terminal regions corresponding, respectively, to the residues 290- <dig>  and residues 404- <dig> .
phylogenetic analysis using amino acid sequences clustered the transposases of paracoccidioides into  <dig> major groups, one carrying trema, b, c, d, e and f elements, and the other harboring tremh and g elements .
this analysis confirms the proximity between trema, tremb and tremc elements  and tremd and treme elements .
tremf elements, although clustered with the trem elements above, are divergent and located in a distinct branch.
based on phylogenetic reconstructions , tremg and tremh seem to form a cluster completely separated from other trem elements.
both groups contain tc1/mariner transposases that are also present in other fungi, e.g.
a. nidulans, a. fumigatus and a. oryzae.
our analysis suggests that trem elements of the different paracoccidioides genomes were derived from a single ancestral sequence present before they diverged from one another.
at a later point in the evolution of this fungus, transposases split into  <dig> separate lineages, giving rise to the family composed of trema, b, c, d, e and f and the other consisting of tremh and g.
distribution and genomic environment of trem elements
to characterize the insertion target sites and preferences in paracoccidioides genomes in more detail, junction fragments between the transposon and the genome were analyzed.
trem elements, like other tc1/mariner transposons, integrate into a ta dinucleotide, causing target-site duplication of the ta sequence at the tir boundary.
additional file  <dig> lists  <dig> independent insertion events identified in the  <dig> paracoccidioides genomes, demonstrating integration of the transposon into a ta dinucleotide within the fungal genome and validating genuine transposition.
analysis of the genomic location of each trem element insertion showed that they were dispersed across the genomes.
they did not, however, appear to be completely random .
genome sequences from isolate pb <dig>  were assembled in  <dig> supercontigs .
the  <dig> hits similar to dna transposons  were found along  <dig> of the  <dig> supercontigs of pb <dig>  of these  <dig>   <dig> contain trem elements , which account for  <dig> % of the genome of isolate pb <dig> .
in the genome of pb <dig> , there are  <dig> hits similar to dna transposons  distributed in  <dig> of the  <dig> supercontigs, and trem elements  were found in  <dig> supercontigs of this isolate , covering  <dig> % of the genome.
in isolate pb <dig> , the  <dig> hits showing similarity to tes  were distributed along  <dig> supercontigs , and trem elements accounted for  <dig> % of the genome .
the above results suggest that trem elements do not integrate in a completely random fashion.
in an extended survey, the genomic context in which trem elements were inserted was examined in  <dig> kb region of supercontigs  <dig>  and  <dig>  of isolate pb <dig> .
in supercontig  <dig>  this particular region has a high density of dna transposons ,  <dig> of which belong to the trem family  .
in addition to the  <dig> trem elements,  <dig> genes  and  <dig> pseudogenes are present.
among the pseudogenes there is an unclassified pogo-like putative dna transposon.
in supercontig  <dig>  we found  <dig> trem elements ,  <dig> genes  and  <dig> pseudogenes.
the analysis showed that trem elements target a wide variety of chromosomal positions and some appearing to be clustered in several regions of the genome.
southern blot and chromoblot hybridization analyses
southern blot hybridization of paracoccidioides genomic dna with trema, tremb and treme probes revealed multiple bands, suggesting that multiple copies were inserted with polymorphic distribution .
differences between the hybridization profiles obtained with the same trem probe confirm the existence of different copy numbers and distribution of the element in the isolates.
no hybridization signal was observed in isolate pb <dig>  with probes trema or tremb.
treme probe hybridized with  <dig> well-defined bands in pb <dig> isolate, and weakly with a  <dig>  kb band in pb <dig> isolate .
probe trema hybridized with  <dig> of the  <dig> chromosomal bands observed in pb <dig> and with  <dig> of the  <dig> in pb <dig>  chromoblot hybridization also confirmed the absence of trema and tremb in isolate pb <dig> .
the p. brasiliensis synteny map shows the location of  <dig> scaffolds in the  <dig> chromosomes of isolate pb18: http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/chromomap.html.
trema and tremb elements are present in  <dig> chromosomes, but are not found in chromosome  <dig>  in accordance with their karyotype profiles .
pcr amplification of trem elements in paracoccidioides isolates of distinct phylogenetic origin
tirs of trem elements consist of two  <dig> bp direct repeats similar to those found in dna transposons of other fungi, namely aft <dig> and tan <dig> .
the repeats are completely conserved except for the tirs of the elements tremb and tremh, in which one substitution was detected .
pcr screening using primers derived from tirs of each trem element was performed on genomic dna from  <dig> clinical isolates, including those from the fgi genome project, and one environmental isolate .
ap.
brasiliensis isolates used in the genome sequencing project - broad institute mit and harvard.
bpcm - paracoccidioidomycosis
cphylogenetic species grouped in accordance with matute et al.
<cit> d, carrero et al.
<cit> e, teixeira et al.
<cit> f and figure  <dig> g.
hisolate was also used by morais et al.
<cit>
irevised by puccia et al.
<cit> .
of the  <dig> isolates,  <dig> had been previously genotyped by sequencing and their phylogenetic relatedness was known; the phylogenetic relatedness of the remaining  <dig> isolates -  <dig> brazilian  and one argentinean  which had not been included in previous sequencing and multi-locus studies - was unknown .
previous work using rapd for typing p. brasiliensis indicated that the employment of certain primers and conditions allowed discrimination of isolates with very low genetic similarity and clustering of the most similar ones  <cit> .
we considered that performing rapd with these highly discriminatory primers would allow us to build a dendrogram in which the genetically most distant branches would correspond to the species level, p. brasiliensis and p. lutzii , clustering isolates corresponding to the phylogenetic species s <dig> and ps <dig>  an assumption that would uncover the phylogenetic relatedness of the  <dig> unknown isolates .
the  <dig> isolates submitted to rapd analysis were classified as belonging to  <dig> of the  <dig> known phylogenetic lineages: s <dig> and ps <dig>  or pb01-like   <cit>  .
analysis of the dendrogram showed  <dig> major clusters with a low coefficient of similarity , the first corresponding to p. lutzii .
the second branch comprised all  <dig> p. brasiliensis isolates showing  <dig> internal clusters , the first including  <dig> s <dig> and the second clustering  <dig> ps <dig> isolates   <cit> .
the p. brasiliensis isolates penguin and pb <dig>  although previously defined as s <dig> , did not group with the s <dig> isolates but appear as  <dig> branches each with only one strain.
it is interesting that the isolates penguin and pb <dig> are from uruguay and argentina, while the majority of the other isolates are brazilian .
a fungal associated feature that has been reported to correlate by rapd branching or clustering of paracoccidioides isolates refers to the isolate's geographic origin  <cit> .
pcr analysis performed with primers corresponding to the tirs produced amplicons with sizes compatible with the trem elements described above, confirming the results obtained in the genomic analysis .
to confirm the pcr specificity, the amplicons were hybridized with probes containing internal regions of each trem element .
as expected, trema was amplified in pb <dig> and pb <dig>  as well as in other isolates phylogenetically related to them, except for penguin and pb <dig> isolates.
a similar amplification pattern was found for tremb in all ps <dig> and s <dig> isolates, except for isolate pb <dig>  trema and tremb were not found in pb01-like isolates.
tremc was found in all isolates.
a strong hybridizing  <dig>  kb band was detected in the pb01-like isolates.
two bands  were amplified in the ps <dig> and s <dig> isolates; however, only the largest ones hybridized to the tremc probe.
tremd was detected in all s <dig> isolates but not in the pb01-like ones.
treme was amplified in pb01-like isolates but not in s <dig> or ps <dig> isolates.
a  <dig>  kb tremf amplicon was detected in ps <dig> and s <dig> isolates.
an additional hybridizing band of ~ <dig>  kb was detected in  <dig> s <dig> isolates, suggesting the presence of degenerate copies of tremf.
in the pb01-like isolates, a smaller pcr product was observed, but it did not hybridize with the tremf probe.
the element tremh was amplified in all isolates we analyzed.
because of low copy-number of tremh in pb01-like isolates, a longer exposure of the autoradiogram was needed to visualize the hybridizing bands.
a strong hybridizing  <dig>  kb band was seen in ps <dig> and s <dig> isolates.
an additional hybridizing band of ~ <dig>  kb was detected in  <dig> s <dig> isolates, suggesting the presence of degenerate copies of tremh.
transcription of trema and b elements
to isolate trem transcripts, cdnas from yeast forms were synthesized by rt-pcr using primers from trema and tremb shown in additional file  <dig>  the rt-pcr products were separated by electrophoresis , purified from the agarose gel and cloned into ta-vectors for sequencing.
transcripts of length corresponding to the unit size of the elements were amplified for pb <dig> and pb <dig> , but not for pb <dig> .
recombinant cdna clones from isolate pb <dig> were sequenced and found to be homologous to trema  and tremb , confirming that these elements are transcribed.
in conclusion, trem elements are not silent in the genome.
discussion
we have carried out the first systematic search for dna transposons in the genomic sequence of the dimorphic pathogenic fungus paracoccidioides, isolates pb <dig> , pb <dig> and pb <dig>  .
the computational search strategy identified approximately  <dig> putative transposon sequences  in the genome of the  <dig> isolates, signaling the occurrence of class ii tes in the genus paracoccidioides.
we identified  <dig> families of transposons of the tc1/mariner superfamily, based on the analysis of transposases and their associated tirs and tsds  <cit> .
with the exception of tremg, in which the tirs could not be defined, trem elements are typical tc1/mariner transposons exhibiting the tir-transposase-tir structure.
tc1/mariner superfamily is probably the most widely distributed transposon family in nature, being frequently found in the genomes of filamentous fungi  <cit> .
however, there have to date been no descriptions of dna transposons in dimorphic fungi.
however, dna transposons in the genus aspergillus , phylogenetically related to the dimorphic fungi, have already been identified  <cit> .
trem elements represent a significant proportion of the genome - around 1% of the genome of p. brasiliensis  and  <dig> % of p. lutzii  - indicating their successful proliferation in the genome.
it is noteworthy that the majority  of putative paracoccidioides dna transposons correspond to degenerated sequences  that are difficult to classify; and were not included in this description.
among the remaining sequences, it may be possible to find elements belonging to the mites , a category of small, nonautonomous dna transposons  carrying preserved tirs found in various organisms, including fungi  <cit> .
of the  <dig> trem copies,  <dig>  may be potentially autonomous active elements, as judged by the presence of intact tirs and orfs coding for transposases.
copies with truncated orfs but preserved tirs may be mobilized in trans by the enzyme coded by an intact copy  <cit> .
hth and dde domains were preserved in the potentially active trem elements.
as previously observed in fungi, the glutamyl residue of the dde domain was substituted by an aspartyl residue   <cit> .
the currently most known eukaryotic cut-and-paste transposons are dde-superfamilies, piggybac and tc1/mariner being the only  <dig> superfamilies encoding ddd-transposases.
conservation of the triad signature  in tc1/mariner transposons may reflect a catalytic mechanism similar to that described for the dde motif, which probably coordinate divalent metal ions to promote catalysis of dna cleavage and ligation.
the cenpb domain was found in trema, tremb and tremf elements.
this domain has been identified in mammalian centromeric proteins and eukaryotic transposases, but not in any archaeal, bacterial or plant proteins.
it may be implicated in the recognition of the terminal inverted repeats of transposons.
the functional similarity between cenpb domains could be the result of domestication of transposons by the host genome to act as its own genes or regulatory elements  <cit> .
the n-terminal hth domain, also found in the truncated elements tremc and treme, has similarity with the "pipsqueak" dna-binding domain of drosophila, a family of eukaryotic proteins that recognize and bind to dna sequences  <cit> .
the terminal inverted repeats were identified in  <dig> of the  <dig> trem elements.
the inverted regions had sizes compatible with those of other elements belonging to the tc1/mariner superfamily described in fungi, and also had a  <dig> bp internal direct repeat, as previously reported  <cit> .
it was suggested that this direct repetition could be the recognition and binding of the hth and cenpb transposase domains  <cit> .
an interesting finding was the presence of conserved cx2cx4hx4c  motifs indicating zinc finger  domains in the c-terminal ends of transposases of at least  <dig> trem elements .
the zf, which is present in a wide range of proteins, forms a structure stabilized by co-ordination of a divalent zinc cation , with cysteine or histidine residues as ligands.
zf are structurally diverse and present among proteins with a broad range of cellular functions, such as replication and repair, transcription and translation, metabolism and signaling, cell proliferation and apoptosis.
krishna et al.
<cit> , who described  <dig> fold groups of zf, discussed their similarities and differences with functional implications.
the fold group  <dig> consists of  <dig> families: c2h <dig> fingers and iap  domains.
iap contains a cchc pattern that coordinates a zinc ion, a pattern similar to that found at the c-terminal region of trem elements transposases.
the zf is responsible for binding to dna or rna in some proteins, or for the protein-protein interaction in others.
ohta et al.
<cit>  described transposases encoded by transposable elements of the insertion sequences  <dig>  of escherichia coli as having n-terminal regions containing a c2c <dig> zf motif.
they showed that the zf motif, as well as the hth motif, is essential for transposition promoted by is1transposase because of their involvement in binding specifically to tirs.
is <dig> transposases with an amino acid substitution in the hth or zf motif lose the ability to promote transposition, indicating that  <dig> domains are responsible for tir-specific dna binding in promoting transposition.
it is likely that the 2-domain structure is required for binding to tirs, not only in is1transposase, but also in transposases encoded by some other transposable elements, in order to place a catalytic domain of transposases in a region near the tir end, where strand transfer reactions occur in transpososomes.
these observations suggest the possibility that transposases encoded by some elements have  <dig> domains involved in the tir-specific dna-binding.
in eukaryotes, a drosophila transposable element p encodes a transposition inhibitor that has a cchc region forming a zf that plays an important role in the binding to the tir sequences at the termini of the p element  <cit> .
when transposons move along the genome, footprints known as tsds  can be formed that flank the element.
most transposon superfamilies may have a characteristic tsd with a specific size and sequence  <cit> .
tsds were found in  <dig> out of the  <dig> trem elements.
it was not possible to determine the tirs in tremg and consequently find the flanking tsd.
the copies of tremh are truncated and probably inactivated, resulting in loss or alteration of the tsd, consequently preventing its identification.
as the dna transposons of the tc1/mariner superfamily have the dinucleotide ta as their insertion site, they can be inserted within virtually any genomic region.
however, regional preferences have been observed for many elements which are inserted in specific regions of the genome  <cit> .
the comparison of the number and identity of nested insertions within individual transposons has been used to estimate the relative ages of transposons.
it is reasonable to assume that the older elements are subjected to interruptions by more recently active elements.
older elements should therefore have higher proportions of nested insertions than younger elements.
assuming that dna transposon insertions occur randomly throughout the genome, there should be a higher proportion of nested insertions for older trem elements than for younger ones.
the following evidence could support the hypothesis of recent transposition and expansion of trem elements in the genome:  the absence of nested trem copies inside other transposons;  the presence of intact open reading frames; and  the presence of highly similar copies in some trem elements.
analysis of southern blots and chromoblots of genomic dna from isolates from  <dig> different species, p. lutzii  and p. brasiliensis , corroborates the in silico data in relation to the presence and distribution of trem elements in these genomes.
trem elements do not appear to have been acquired simultaneously by the paracoccidioides sequenced genomes, as indicated by finding of different copy-numbers among trem families.
the elements tremc and tremh are present in a large number of copies, most of which are truncated elements with stop codons.
unlike tremc and tremh, tremd is only present in  <dig> copies,  <dig> of which have integral orfs, just in isolate pb <dig> .
this pattern is compatible with a recent insertion in the genome, as tremd would not yet have been inactivated by the genome defense system.
several recent studies have proposed the existence of different phylogenetic species in the taxon p. brasiliensis.
matute et al.
<cit>  analyzed the genetic structure of  <dig> p. brasiliensis isolates, concluding that they can be grouped into  <dig> distinct phylogenetic species: s <dig> , ps <dig>  and ps <dig> .
in a study of  <dig> p. brasiliensis isolates,  <dig> of which had been included in the above study, carrero et al.
<cit>  reached a similar conclusion for all the isolates except isolate pb <dig>  which they suggested was a new phylogenetic species in the genus paracoccidioides.
recently, teixeira et al.
<cit> , analyzing  <dig> isolates of the fungus, found that  <dig> were genotypically similar, belonging to the pb01-like group.
they estimated that the s1/ps2/ps <dig> species clade and the pb01-like new species, for which the name p. lutzii was proposed, shared a common ancestor approximately  <dig> million years ago.
we used pcr to analyze the distribution of trem elements in  <dig> clinical isolates and one environmental isolate formerly known as p. brasiliensis.
previous reports using rapd for typing p. brasiliensis indicated that the use of certain primers and conditions allowed the discrimination of isolates with very low genetic similarity clustering the most similar ones.
the primers chosen discriminated of p. brasiliensis isolates into groups with very low genetic similarity indexes   <cit> .
probably for this reason our rapd analysis resulted in the clustering of isolates previously known to belong to a certain phylogenetic species , which could be a reference for the characterization of those of unknown genetic relatedness .
the  <dig> isolates consisted of the sequenced pb <dig> and  <dig> pb01-like isolates   <cit> ;  <dig> isolates previously classified as ps <dig> ; and  <dig> isolates identified as s <dig> .
ps <dig>  the fourth known phylogenetic species reported to be exclusively in colombia was not represented  <cit> .
the isolates were grouped in accordance with matute et al.
<cit> , carrero et al.
<cit> , teixeira et al.
<cit>  and the results of rapd analysis   <cit> .
isolates penguin and pb <dig> are known to be p. brasiliensis  but do not cluster with the other s <dig> isolates .
these isolates come from different geographic regions, uruguay and argentina, while the majority is brazilian .
it is interesting that a fungal associated feature that has been reported to correlate by rapd branching or clustering of paracoccidioides refers to the isolate's geographic origin  <cit> .
the non-grouping of all s <dig> isolates would be explained by the fact that multilocus sequencing studies employ nuclear genes while rapd would arbitrarily reveal polymorphic regions along the genomes, including intergenic and/or repetitive sequence.
the dna transposons tremc and tremh were identified in all the isolates tested, indicating that these elements would have already been present in a hypothetical common ancestor rather than have been acquired horizontally after the  <dig> phylogenetic species diverged from one another.
in contrast, treme was only found in pb <dig> and pb01-like isolates, and trema, tremb, and tremf were found in the s <dig> and ps <dig> isolates, while the element tremd was almost exclusively found in s <dig> isolates, suggesting that these dna transposons could have been acquired horizontally after separation of the  <dig> phylogenetic species.
although tremd and treme share approx 70% similarity at the amino acid level, they were found exclusively in the s <dig> and pb01-like isolates, respectively, which are phylogenetically distant.
this suggests that these elements would have been acquired by horizontal transfer after the split between s1/ps <dig>  and pb01-like species .
this hypothesis is supported by the finding of hypothetical protein sequences of a. capsulatum in genbank  that yielded an e-value of  <dig>  for the elements tremd and treme, respectively .
however, the absence of tremd and treme could also be explained by the loss of these elements in some strains.
transcription of trema and tremb suggests that they could be active elements.
when transposing, the elements generate double-strand dna breaks, which are repaired by the cellular machinery  <cit> .
hence, in addition to the direct effects of transposition, such as gene inactivation, recombination events promoted by repair of the double strand would also take place.
transposition of these elements could be a factor involved in the genetic variability observed in the p. brasiliensis species complex.
the identification of active elements would serve as a basis for the development of mutational tools, as transposons could be efficient vectors for modifications.
CONCLUSIONS
the strategy developed in the present work proved effective in identifying and characterizing dna transposons in the newly generated sequence assemblies of isolates of  <dig> phylogenetic species of the p. brasiliensis complex.
the most intact insertions allowed the classification of  <dig> new families and a new subfamily of elements  that share identity with the tc1/mariner superfamily, distinct lineages not yet identified in dimorphic fungi.
three full-length, potentially functional autonomous elements were characterized .
further studies to show their active transposition would be interesting.
the phylogenetic analysis supports the hypothesis that trem families derived from a single ancestral sequence and split into  <dig> lineages .
the occurrence of trem families is unequal in the genomic sequence of the isolates pb <dig>  pb <dig> and pb <dig> and experimental evidence from the other  <dig> typed isolates indicated that some trem families  were shared while other trem families were harbored exclusively by pb01-like species   or s1/ps <dig> species  .
it would be interesting to verify the occurrence of distinct trem families in a larger number of p. lutzii and p. brasiliensis isolates from different origins , and from different geographic regions, also including the phylogenetic species ps <dig>  not represented here, in such a way as to expand and explore the potential of these newly presented genetic agents as sources of phylogenetic and epidemiological knowledge.

 the thermodimorphic fungus paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic endemic disease that affects at least million people in latin america. infection probably occurs as a result of inhalation of conidia that subsequently transform into yeast forms within the lung, resulting in asymptomatic infection or infection that can progress to acute and chronic clinical forms of the disease. paracoccidioidomycosis is the 8th most common cause of death due to chronic/recurrent infections and parasitic diseases in brazil. the teleomorph stage of this fungus is unknown, but analysis of the large rdna subunit classified it as an ascomycete, order onygenales, family onygenaceae, phylogenetically close to ajellomyces, a genus that harbors other human dimorphic fungal pathogens, specifically histoplasma capsulatum and blastomyces dermatitidis. molecular analysis by random amplification of polymorphic dna, restriction fragment length polymorphism and electrophoretic karyotyping showed extensive genetic variability among distinct isolates. rapd, a technique popular for its simplicity, has been useful in determining the existence of genetically distinct p. brasiliensis groups, their relationship with geographic distribution, and the similarity among clinical, animal and environmental samples. however, the information has been difficult to compare. gene polymorphism studies were pioneered by characterization of pbgp polymorphism, a gene encoding the fungus immunodominant antigen, in a sample of isolates. by comparing sequenced pcr fragments from the whole gene these authors found informative substitution sites, mostly in exon which defined 5- genotypes. the maximum-likelihood phylogenetic tree generated with these sequences clearly reflected the presence of specific genetic groups in the species. subsequently, multilocus genealogy studies demonstrated evolutionary lineages identifying the occurrence of cryptic phylogenetic species that were morphologically indistinguishable by following the criteria of genealogical concordance and non-discordance which is used to identify the reduction of gene flow between groups of individuals due to geographic or reproductive isolation barriers. the pbgp locus, whose characteristic genotypes have recently been reviewed, was the most polymorphic and informative in these studies. matute et al. analyzed the genetic structure of p. brasiliensis isolates and concluded that they could be grouped into distinct phylogenetic species: s, ps and ps. in a study of p. brasiliensis isolates, of which had been included in the above study, carrero et al. came to a similar conclusion for all the isolates, with the exception of isolate pb which they suggested was a new phylogenetic species in the genus paracoccidioides. recently, teixeira et al., analyzing isolates of the fungus, found that of them were genotypically similar, belonging to the pb01-like group. they estimated that the s1/ps2/ps species clade and the pb01-like new species, for which the name paracoccidioides lutzii was proposed, shared a common ancestor approximately million years ago. these studies suggested that the genus paracoccidioides consists of at least different, previously unrecognized phylogenetic species: p. brasiliensis s, ps and ps, and a 4th quite distinct lineage comprising fungal isolates formerly referred to as pb01-like, now proposed to be a new species, p. lutzii. the multilocus sequence analysis also supported recombination in nature in p. brasiliensis and p. lutzii, indicating the presence of sexual reproduction. thanks to the broad institute fungal genome initiative project, the complete genome of representative isolates from p. brasiliensis s, ps and pb like have recently been made available at the site: http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis thus allowing further and specific comparisons among isolates. previous local projects, however, contributed to the construction of large expressed sequence tag databases of pb and pb. transposable elements represent a substantial fraction of eukaryotic genomes that have been found in virtually all species investigated to date. they are abundant in fungi and represent 3-20% of the genomes of many filamentous fungi. tes can produce a variety of effects in genomes, such as gene inactivation or modification, chromosome breakage, and genome recombination or rearrangement, thereby generating plasticity. karyotyping and gene mapping indicated the occurrence of chromosomal rearrangements, which are associated with the presence of repetition and te in fungal genomes. tes can be grouped into classes according to their transposition mechanism. class i consists of retrotransposons that transpose via an rna intermediate that is reverse-transcribed into dna by a te-encoded reverse transcriptase. class ii elements, also called dna transposons, move via a dna intermediate. because of the abundance and diversity of tes described in the eukaryotic genomes, a unified classification system for eukaryotic tes has been proposed by different groups. dna transposons can be divided into subclasses: the first comprises the elements that transpose via the classical "cut-and-paste" mechanism; the second comprises elements that transpose by replication involving the displacement of only one strand; and in the third, transposons move by a mechanism called "rolling-circle". subclass dna transposons require the cleavage of both dna strands for the transposition and have orders - tir and crypton. six superfamilies of the order tir from subclass have been identified in fungi: tc1/mariner, hat, mutator, transib, pif-harbinger and cacta. tir transposases contain a signature active site motif that consists of acidic amino-acids and forms the catalytic pocket of the enzyme responsible for cleaving dna strands. crypton, the only superfamily representative of the second order, has only been found to date in fungi and, unlike any previously described transposons, encodes a putative tyrosine recombinase instead of a classical transposase. this recombinase lacks an rt domain, which suggests that transposition occurs via a dna intermediate. it does not contain long repeat sequences, but is bordered by short direct repeats that may have been generated by its insertion into the host genome by recombination. cryptons may be excised from the host genome by tyrosine recombinase, generating a circular, extrachromosomal, double-stranded dna molecule, for which cleavage of both dna strands is required. elements from subclasses and have only one strand cleaved during transposition and have also been identified in fungi, but the tc1/mariner superfamily is probably the most prevalent in these organisms. transposons contain terminal inverted repeats, which are recognized by the pipsqueak helix-turn-helix motif in the transposase domain. each subclass dna transposon superfamily is characterized by a specific transposase core that is different from those of other superfamilies. upon insertion, dna transposons generate target site duplications with lengths that are relatively well conserved among superfamily members. tes have been poorly characterized in p. brasiliensis. transcriptome analysis indicated that ty-like retroelements were among the most abundant ests in p. brasiliensis. nascimento et al., searching for microsatellites that could be potentially useful as molecular markers, reported that a small number of them were located in transposons. the focus of our work has been the characterization of dna transposons and their derivatives in the genus paracoccidioides. to this end we searched for dna transposons in the paracoccidioides genomic sequence database http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis. we have discovered new families of dna transposons and one new subfamily of mariner-1_af belonging to the tc1/mariner superfamily. remarkably, some trem elements appear that may be active in the genomes of representative isolates of different paracoccidioides phylogenetic lineages. the finding of active autonomous dna transposons in p. brasiliensis may have implications for an understanding of the evolutionary processes underlying the diversification of this group. furthermore, transposons are efficient vectors for introducing foreign dna into cells.  occurrence of dna transposons in the genus paracoccidioides a survey of the p. brasiliensis pb functional and differential genome project annotated est database for sequences similar to transposase sequences found contigs that contain open reading frames encoding proteins of and amino acids, respectively, with similarity to the conserved hth dna binding domain of the transposase of the aft dna transposon in aspergillus fumigatus. these sequences were amplified from p. brasiliensis genomic dna, their identities confirmed by sequence analysis, and then used as probes in southern blot hybridization with genomic dna of different p. brasiliensis isolates. this revealed the presence of multiple copies of dna transposons dispersed throughout the genome of different isolates. rt-pcr analysis of total rna of the isolates pb, pb and pb confirmed transcription of the putative elements. the transposon sequence from each est contig was used as a query in a modified blastn search against the whole genome sequence assemblies of all paracoccidioides genomes and combined with the strategy for identification of tirs, what allowed the artemis annotation tool to be used to identify transposons. adna transposase sequences from the p. brasiliensis transcriptome were used to perform queries in a modified blastn search in the genome of isolates pb pb and pb and combined with a strategy for identification of tirs. belements assigned to a dna transposon superfamily according to the standard principles of the te classification proposed by wicker et al.. csequences that do not meet all the criteria proposed by wicker et al.. they are highly defective elements, with lower conservation of the typical transposase domains. we also searched the paracoccidioides genomes for cryptons, rolling-circle dna transposons and self-synthesizing dna transposons, using consensus sequences for these elements, no significant matches were found. in the genomic sequence of pb, only dna transposon families have been classified, corresponding to of the hits found in this isolate. in the set of elements for pb full-length potentially functional autonomous elements coding for putative transposases with well-defined tirs were classified. the copies of the elements found in pb totaled hits, or % of the hits, similar to dna transposons in this isolate. of the hits registered in pb were identified and correlated with tc1/mariner elements. tc1/mariner superfamily in the genus paracoccidioides we identified dna transposon elements in paracoccidioides that share 65-80% identity at the nucleotide level with tc1/mariner transposons deposited in repbase at the genetic information research institute. like the tc1/mariner transposons, of these elements are flanked by ta dinucleotide. because of their similarity to the tc1/mariner superfamily and the fact that most of them insert specifically in ta target sites, these elements were given the name trem. the completeness of transposons was confirmed by the presence of tirs at both ends, followed by multiple sequence alignment to well-studied examples. incomplete sequences were not included in the comparative analyses shown in figure we examined insertion loci of trem elements, of which are flanked by well-defined tirs, while the remaining sequences, including all tremg elements, are not flanked by tirs. atsd - target site duplication. ta dinucleotide. brepeat masking - results of the analysis performed with the repeat masking script using the girinst http://www.girinst.org/repbase database and percentage similarity. csize - total length of element in base pairs dprotein - length of transposase in amino acids. some elements contain or orfs encoding different proteins. etirs - length of terminal inverted repeats in base pairs. fcopy number - number of te copies found in the genome of the p. brasiliensis isolates. the number of elements with potentially functional orfs is shown in parentheses. gn - the total number of tc1/mariner transposons identified in p. brasiliensis. there are copies of trema in the pb isolate, which contain a full-length orf and which are truncated, and copies in the pb isolate, which have an intact orf. trema sequences were aligned to build an bp consensus containing a single orf that encodes a amino acid protein with conserved domains characteristic of transposases - hth, cenpb and dde. the trema tirs are bp long and have a bp internal direct repeat. analysis of trema with the repeatmasker script in the girinst database http://www.girinst.org/repbase revealed a similarity of only 66% with the a. fumigatus mariner-1_af transposon, suggesting that it may be a new dna transposon family. we found copies of tremb, in the genome of the pb isolate and in pb. however, only copies showed intact orfs, which were used to generate a bp consensus sequence flanked by bp tirs. this orf encodes a aa transposase with conserved domains. analysis using repeatmasker gave an index of 65% similarity with the mariner-1_af. it is interesting that tremb sequences carrying truncated orfs are flanked by intact tirs. tremc is the most abundant dna transposon, having truncated copies dispersed throughout the genomes. the longest sequences were aligned to generate a bp consensus flanked by bp tirs. it contains short orfs, which encode the hth and dde domains. the central region of the element shows similarity with a. fumigatus transposases, and analysis by repeatmasker revealed 66% similarity with mariner-2_ao in aspergillus oryzae. the tremc shown in figure is based on the element's most conserved sequences. tremd is present in a small number of copies, exclusively in the pb isolate. the consensus sequence has bp and harbors a bp orf flanked by bp tirs. the orf codes for a aa transposase carrying the hth and dde domains. repeatmasker analysis showed 68% similarity with mariner-1_af in the central region, and 80% similarity with the tirs, suggesting that tremd is related to or belongs to this family. the consensus sequence for the treme transposon, found exclusively in the pb isolate, is bp long, flanked by bp tirs. it includes orfs: one has bp and codes for a aa transposase with the hth and dde domains; and the second encodes a aa protein similar to a hypothetical protein from ajellomyces capsulatum. treme showed a 65% similarity with the mariner-1_af transposon. with its bp consensus sequence, tremf is the longest of the elements described in this study and the third most abundant. the consensus sequence is flanked by bp tirs and contains orfs: the first is bp long and codes for a aa protein harboring the conserved domain cenpb; the second extends from position nt to and codes for a aa dde-domain-harboring protein. the results of repeatmasker analysis revealed a 67% similarity with mariner-9_an in emericella nidulans, which is a non-autonomous dna transposon belonging to the pogo clade. thus, tremf could represent a new transposon family. tremg, the 7th tc1/mariner element we identified, has copies in pb and in pb. all copies are truncated, flanked by highly divergent and irregular sequences. the central core of some elements has an orf coding for a aa protein carrying the dde domain. unfortunately, it was not possible to determine the precise boundary of this element because of the divergence among the flanking sequences. repeatmasker analysis showed a 66% similarity with the mariner-5_af in a.fumigatus, a dna transposon of the tc clade. for this reason, this element was classified as a tc1/mariner transposon. tremh is present in the isolates, with copies in pb in pb and in pb. of the copies of this element, are flanked by bp tirs; however, it was not possible to define its target site. we used copies that had the best sequence conservation and encoded the dde motif to rebuild the bp consensus sequence. repeatmasker analysis showed a 66% similarity with the mariner3_ao in a.oryzae. the element copy contains an orf coding for a aa protein carrying the dde motif, similar to that in the transposon in talaromyces stipitatus. in this region, strictly conserved acidic amino acids - d, d and d - were found, and these probably constitute the dde catalytic motif in tremh. since the transposase activity in many dna transposons relies upon the catalytic activity of the dde domain, we suggest that tremh is a tc1/mariner-like transposon. phylogenetic analysis the alignment of the predicted sequences corresponding to each of the conserved transposase domains found in dna transposons in paracoccidioides, with the sequences of well-characterized dna transposons of the tc1/mariner superfamily from other species, allows the identification of motifs considered essential for active transposition. some of the paracoccidioides transposases are interrupted by stop codons, minor indels or the absence of a translation initiation codon. the complete catalytic triad dde occurred in of the elements and was characterized by the substitution of the glutamate residue by aspartate to form a d, d, d motif, as previously observed in other fungal transposases. the triad signatures are very similar and can be represented by d10--d122--d in the elements trema, b, d and e; d10--d120--d in the element tremg; and d7--d119--d in the element tremh. in tremf, the first aspartic residue was deleted, but the distance between the second and third aspartate residues was maintained -d125). in tremc, all aspartic residues were absent. in addition to the conserved catalytic triad ddd, transposases contain a number of other highly conserved, group-specific amino acids, such as the hth and cenpb domains. the hth domain was found in trem elements with varying degrees of conservation - 58% identity between the pairs trema/tremb, tremb/tremc and tremd/treme, and 53% identity between trema/tremc. the identities between treme/trema and treme/tremb were lower than 40%. alignment of the dna-binding domains of the transposases revealed a distinction between trem elements, which may be functionally connected to the tirs of these elements. when the hth domain was compared with that of other fungal transposases, the highest identity index was found between tremd and the tan transposon of aspergillus niger. the cenpb domain was present in of the paracoccidioides transposons. the identity between trema and tremb was %, and between these elements and trem f less than 36%. it was also observed that elements presented conserved cx2cx4hx4c zinc finger domains in the c-terminal ends of their transposases corresponding, respectively, to amino acid residues 531- 528- and 540- in tremc and treme, similar but non-canonical motifs were present at the c-terminal regions corresponding, respectively, to the residues 290- and residues 404-. phylogenetic analysis using amino acid sequences clustered the transposases of paracoccidioides into major groups, one carrying trema, b, c, d, e and f elements, and the other harboring tremh and g elements. this analysis confirms the proximity between trema, tremb and tremc elements and tremd and treme elements. tremf elements, although clustered with the trem elements above, are divergent and located in a distinct branch. based on phylogenetic reconstructions, tremg and tremh seem to form a cluster completely separated from other trem elements. both groups contain tc1/mariner transposases that are also present in other fungi, e.g. a. nidulans, a. fumigatus and a. oryzae. our analysis suggests that trem elements of the different paracoccidioides genomes were derived from a single ancestral sequence present before they diverged from one another. at a later point in the evolution of this fungus, transposases split into separate lineages, giving rise to the family composed of trema, b, c, d, e and f and the other consisting of tremh and g. distribution and genomic environment of trem elements to characterize the insertion target sites and preferences in paracoccidioides genomes in more detail, junction fragments between the transposon and the genome were analyzed. trem elements, like other tc1/mariner transposons, integrate into a ta dinucleotide, causing target-site duplication of the ta sequence at the tir boundary. additional file lists independent insertion events identified in the paracoccidioides genomes, demonstrating integration of the transposon into a ta dinucleotide within the fungal genome and validating genuine transposition. analysis of the genomic location of each trem element insertion showed that they were dispersed across the genomes. they did not, however, appear to be completely random. genome sequences from isolate pb were assembled in supercontigs. the hits similar to dna transposons were found along of the supercontigs of pb of these contain trem elements, which account for % of the genome of isolate pb. in the genome of pb, there are hits similar to dna transposons distributed in of the supercontigs, and trem elements were found in supercontigs of this isolate, covering % of the genome. in isolate pb, the hits showing similarity to tes were distributed along supercontigs, and trem elements accounted for % of the genome. the above results suggest that trem elements do not integrate in a completely random fashion. in an extended survey, the genomic context in which trem elements were inserted was examined in kb region of supercontigs and of isolate pb. in supercontig this particular region has a high density of dna transposons, of which belong to the trem family. in addition to the trem elements, genes and pseudogenes are present. among the pseudogenes there is an unclassified pogo-like putative dna transposon. in supercontig we found trem elements, genes and pseudogenes. the analysis showed that trem elements target a wide variety of chromosomal positions and some appearing to be clustered in several regions of the genome. southern blot and chromoblot hybridization analyses southern blot hybridization of paracoccidioides genomic dna with trema, tremb and treme probes revealed multiple bands, suggesting that multiple copies were inserted with polymorphic distribution. differences between the hybridization profiles obtained with the same trem probe confirm the existence of different copy numbers and distribution of the element in the isolates. no hybridization signal was observed in isolate pb with probes trema or tremb. treme probe hybridized with well-defined bands in pb isolate, and weakly with a kb band in pb isolate. probe trema hybridized with of the chromosomal bands observed in pb and with of the in pb chromoblot hybridization also confirmed the absence of trema and tremb in isolate pb. the p. brasiliensis synteny map shows the location of scaffolds in the chromosomes of isolate pb18: http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/chromomap.html. trema and tremb elements are present in chromosomes, but are not found in chromosome in accordance with their karyotype profiles. pcr amplification of trem elements in paracoccidioides isolates of distinct phylogenetic origin tirs of trem elements consist of two bp direct repeats similar to those found in dna transposons of other fungi, namely aft and tan. the repeats are completely conserved except for the tirs of the elements tremb and tremh, in which one substitution was detected. pcr screening using primers derived from tirs of each trem element was performed on genomic dna from clinical isolates, including those from the fgi genome project, and one environmental isolate. ap. brasiliensis isolates used in the genome sequencing project - broad institute mit and harvard. bpcm - paracoccidioidomycosis cphylogenetic species grouped in accordance with matute et al. d, carrero et al. e, teixeira et al. f and figure g. hisolate was also used by morais et al. irevised by puccia et al.. of the isolates, had been previously genotyped by sequencing and their phylogenetic relatedness was known; the phylogenetic relatedness of the remaining isolates - brazilian and one argentinean which had not been included in previous sequencing and multi-locus studies - was unknown. previous work using rapd for typing p. brasiliensis indicated that the employment of certain primers and conditions allowed discrimination of isolates with very low genetic similarity and clustering of the most similar ones. we considered that performing rapd with these highly discriminatory primers would allow us to build a dendrogram in which the genetically most distant branches would correspond to the species level, p. brasiliensis and p. lutzii, clustering isolates corresponding to the phylogenetic species s and ps an assumption that would uncover the phylogenetic relatedness of the unknown isolates. the isolates submitted to rapd analysis were classified as belonging to of the known phylogenetic lineages: s and ps or pb01-like. analysis of the dendrogram showed major clusters with a low coefficient of similarity, the first corresponding to p. lutzii. the second branch comprised all p. brasiliensis isolates showing internal clusters, the first including s and the second clustering ps isolates. the p. brasiliensis isolates penguin and pb although previously defined as s, did not group with the s isolates but appear as branches each with only one strain. it is interesting that the isolates penguin and pb are from uruguay and argentina, while the majority of the other isolates are brazilian. a fungal associated feature that has been reported to correlate by rapd branching or clustering of paracoccidioides isolates refers to the isolate's geographic origin. pcr analysis performed with primers corresponding to the tirs produced amplicons with sizes compatible with the trem elements described above, confirming the results obtained in the genomic analysis. to confirm the pcr specificity, the amplicons were hybridized with probes containing internal regions of each trem element. as expected, trema was amplified in pb and pb as well as in other isolates phylogenetically related to them, except for penguin and pb isolates. a similar amplification pattern was found for tremb in all ps and s isolates, except for isolate pb trema and tremb were not found in pb01-like isolates. tremc was found in all isolates. a strong hybridizing kb band was detected in the pb01-like isolates. two bands were amplified in the ps and s isolates; however, only the largest ones hybridized to the tremc probe. tremd was detected in all s isolates but not in the pb01-like ones. treme was amplified in pb01-like isolates but not in s or ps isolates. a kb tremf amplicon was detected in ps and s isolates. an additional hybridizing band of ~ kb was detected in s isolates, suggesting the presence of degenerate copies of tremf. in the pb01-like isolates, a smaller pcr product was observed, but it did not hybridize with the tremf probe. the element tremh was amplified in all isolates we analyzed. because of low copy-number of tremh in pb01-like isolates, a longer exposure of the autoradiogram was needed to visualize the hybridizing bands. a strong hybridizing kb band was seen in ps and s isolates. an additional hybridizing band of ~ kb was detected in s isolates, suggesting the presence of degenerate copies of tremh. transcription of trema and b elements to isolate trem transcripts, cdnas from yeast forms were synthesized by rt-pcr using primers from trema and tremb shown in additional file the rt-pcr products were separated by electrophoresis, purified from the agarose gel and cloned into ta-vectors for sequencing. transcripts of length corresponding to the unit size of the elements were amplified for pb and pb, but not for pb. recombinant cdna clones from isolate pb were sequenced and found to be homologous to trema and tremb, confirming that these elements are transcribed. in conclusion, trem elements are not silent in the genome. discussion we have carried out the first systematic search for dna transposons in the genomic sequence of the dimorphic pathogenic fungus paracoccidioides, isolates pb, pb and pb. the computational search strategy identified approximately putative transposon sequences in the genome of the isolates, signaling the occurrence of class ii tes in the genus paracoccidioides. we identified families of transposons of the tc1/mariner superfamily, based on the analysis of transposases and their associated tirs and tsds. with the exception of tremg, in which the tirs could not be defined, trem elements are typical tc1/mariner transposons exhibiting the tir-transposase-tir structure. tc1/mariner superfamily is probably the most widely distributed transposon family in nature, being frequently found in the genomes of filamentous fungi. however, there have to date been no descriptions of dna transposons in dimorphic fungi. however, dna transposons in the genus aspergillus, phylogenetically related to the dimorphic fungi, have already been identified. trem elements represent a significant proportion of the genome - around 1% of the genome of p. brasiliensis and % of p. lutzii - indicating their successful proliferation in the genome. it is noteworthy that the majority of putative paracoccidioides dna transposons correspond to degenerated sequences that are difficult to classify; and were not included in this description. among the remaining sequences, it may be possible to find elements belonging to the mites, a category of small, nonautonomous dna transposons carrying preserved tirs found in various organisms, including fungi. of the trem copies, may be potentially autonomous active elements, as judged by the presence of intact tirs and orfs coding for transposases. copies with truncated orfs but preserved tirs may be mobilized in trans by the enzyme coded by an intact copy. hth and dde domains were preserved in the potentially active trem elements. as previously observed in fungi, the glutamyl residue of the dde domain was substituted by an aspartyl residue. the currently most known eukaryotic cut-and-paste transposons are dde-superfamilies, piggybac and tc1/mariner being the only superfamilies encoding ddd-transposases. conservation of the triad signature in tc1/mariner transposons may reflect a catalytic mechanism similar to that described for the dde motif, which probably coordinate divalent metal ions to promote catalysis of dna cleavage and ligation. the cenpb domain was found in trema, tremb and tremf elements. this domain has been identified in mammalian centromeric proteins and eukaryotic transposases, but not in any archaeal, bacterial or plant proteins. it may be implicated in the recognition of the terminal inverted repeats of transposons. the functional similarity between cenpb domains could be the result of domestication of transposons by the host genome to act as its own genes or regulatory elements. the n-terminal hth domain, also found in the truncated elements tremc and treme, has similarity with the "pipsqueak" dna-binding domain of drosophila, a family of eukaryotic proteins that recognize and bind to dna sequences. the terminal inverted repeats were identified in of the trem elements. the inverted regions had sizes compatible with those of other elements belonging to the tc1/mariner superfamily described in fungi, and also had a bp internal direct repeat, as previously reported. it was suggested that this direct repetition could be the recognition and binding of the hth and cenpb transposase domains. an interesting finding was the presence of conserved cx2cx4hx4c motifs indicating zinc finger domains in the c-terminal ends of transposases of at least trem elements. the zf, which is present in a wide range of proteins, forms a structure stabilized by co-ordination of a divalent zinc cation, with cysteine or histidine residues as ligands. zf are structurally diverse and present among proteins with a broad range of cellular functions, such as replication and repair, transcription and translation, metabolism and signaling, cell proliferation and apoptosis. krishna et al., who described fold groups of zf, discussed their similarities and differences with functional implications. the fold group consists of families: c2h fingers and iap domains. iap contains a cchc pattern that coordinates a zinc ion, a pattern similar to that found at the c-terminal region of trem elements transposases. the zf is responsible for binding to dna or rna in some proteins, or for the protein-protein interaction in others. ohta et al. described transposases encoded by transposable elements of the insertion sequences of escherichia coli as having n-terminal regions containing a c2c zf motif. they showed that the zf motif, as well as the hth motif, is essential for transposition promoted by is1transposase because of their involvement in binding specifically to tirs. is transposases with an amino acid substitution in the hth or zf motif lose the ability to promote transposition, indicating that domains are responsible for tir-specific dna binding in promoting transposition. it is likely that the 2-domain structure is required for binding to tirs, not only in is1transposase, but also in transposases encoded by some other transposable elements, in order to place a catalytic domain of transposases in a region near the tir end, where strand transfer reactions occur in transpososomes. these observations suggest the possibility that transposases encoded by some elements have domains involved in the tir-specific dna-binding. in eukaryotes, a drosophila transposable element p encodes a transposition inhibitor that has a cchc region forming a zf that plays an important role in the binding to the tir sequences at the termini of the p element. when transposons move along the genome, footprints known as tsds can be formed that flank the element. most transposon superfamilies may have a characteristic tsd with a specific size and sequence. tsds were found in out of the trem elements. it was not possible to determine the tirs in tremg and consequently find the flanking tsd. the copies of tremh are truncated and probably inactivated, resulting in loss or alteration of the tsd, consequently preventing its identification. as the dna transposons of the tc1/mariner superfamily have the dinucleotide ta as their insertion site, they can be inserted within virtually any genomic region. however, regional preferences have been observed for many elements which are inserted in specific regions of the genome. the comparison of the number and identity of nested insertions within individual transposons has been used to estimate the relative ages of transposons. it is reasonable to assume that the older elements are subjected to interruptions by more recently active elements. older elements should therefore have higher proportions of nested insertions than younger elements. assuming that dna transposon insertions occur randomly throughout the genome, there should be a higher proportion of nested insertions for older trem elements than for younger ones. the following evidence could support the hypothesis of recent transposition and expansion of trem elements in the genome: the absence of nested trem copies inside other transposons; the presence of intact open reading frames; and the presence of highly similar copies in some trem elements. analysis of southern blots and chromoblots of genomic dna from isolates from different species, p. lutzii and p. brasiliensis, corroborates the in silico data in relation to the presence and distribution of trem elements in these genomes. trem elements do not appear to have been acquired simultaneously by the paracoccidioides sequenced genomes, as indicated by finding of different copy-numbers among trem families. the elements tremc and tremh are present in a large number of copies, most of which are truncated elements with stop codons. unlike tremc and tremh, tremd is only present in copies, of which have integral orfs, just in isolate pb. this pattern is compatible with a recent insertion in the genome, as tremd would not yet have been inactivated by the genome defense system. several recent studies have proposed the existence of different phylogenetic species in the taxon p. brasiliensis. matute et al. analyzed the genetic structure of p. brasiliensis isolates, concluding that they can be grouped into distinct phylogenetic species: s, ps and ps. in a study of p. brasiliensis isolates, of which had been included in the above study, carrero et al. reached a similar conclusion for all the isolates except isolate pb which they suggested was a new phylogenetic species in the genus paracoccidioides. recently, teixeira et al., analyzing isolates of the fungus, found that were genotypically similar, belonging to the pb01-like group. they estimated that the s1/ps2/ps species clade and the pb01-like new species, for which the name p. lutzii was proposed, shared a common ancestor approximately million years ago. we used pcr to analyze the distribution of trem elements in clinical isolates and one environmental isolate formerly known as p. brasiliensis. previous reports using rapd for typing p. brasiliensis indicated that the use of certain primers and conditions allowed the discrimination of isolates with very low genetic similarity clustering the most similar ones. the primers chosen discriminated of p. brasiliensis isolates into groups with very low genetic similarity indexes. probably for this reason our rapd analysis resulted in the clustering of isolates previously known to belong to a certain phylogenetic species, which could be a reference for the characterization of those of unknown genetic relatedness. the isolates consisted of the sequenced pb and pb01-like isolates; isolates previously classified as ps; and isolates identified as s. ps the fourth known phylogenetic species reported to be exclusively in colombia was not represented. the isolates were grouped in accordance with matute et al., carrero et al., teixeira et al. and the results of rapd analysis. isolates penguin and pb are known to be p. brasiliensis but do not cluster with the other s isolates. these isolates come from different geographic regions, uruguay and argentina, while the majority is brazilian. it is interesting that a fungal associated feature that has been reported to correlate by rapd branching or clustering of paracoccidioides refers to the isolate's geographic origin. the non-grouping of all s isolates would be explained by the fact that multilocus sequencing studies employ nuclear genes while rapd would arbitrarily reveal polymorphic regions along the genomes, including intergenic and/or repetitive sequence. the dna transposons tremc and tremh were identified in all the isolates tested, indicating that these elements would have already been present in a hypothetical common ancestor rather than have been acquired horizontally after the phylogenetic species diverged from one another. in contrast, treme was only found in pb and pb01-like isolates, and trema, tremb, and tremf were found in the s and ps isolates, while the element tremd was almost exclusively found in s isolates, suggesting that these dna transposons could have been acquired horizontally after separation of the phylogenetic species. although tremd and treme share approx 70% similarity at the amino acid level, they were found exclusively in the s and pb01-like isolates, respectively, which are phylogenetically distant. this suggests that these elements would have been acquired by horizontal transfer after the split between s1/ps and pb01-like species. this hypothesis is supported by the finding of hypothetical protein sequences of a. capsulatum in genbank that yielded an e-value of for the elements tremd and treme, respectively. however, the absence of tremd and treme could also be explained by the loss of these elements in some strains. transcription of trema and tremb suggests that they could be active elements. when transposing, the elements generate double-strand dna breaks, which are repaired by the cellular machinery. hence, in addition to the direct effects of transposition, such as gene inactivation, recombination events promoted by repair of the double strand would also take place. transposition of these elements could be a factor involved in the genetic variability observed in the p. brasiliensis species complex. the identification of active elements would serve as a basis for the development of mutational tools, as transposons could be efficient vectors for modifications.  the strategy developed in the present work proved effective in identifying and characterizing dna transposons in the newly generated sequence assemblies of isolates of phylogenetic species of the p. brasiliensis complex. the most intact insertions allowed the classification of new families and a new subfamily of elements that share identity with the tc1/mariner superfamily, distinct lineages not yet identified in dimorphic fungi. three full-length, potentially functional autonomous elements were characterized. further studies to show their active transposition would be interesting. the phylogenetic analysis supports the hypothesis that trem families derived from a single ancestral sequence and split into lineages. the occurrence of trem families is unequal in the genomic sequence of the isolates pb pb and pb and experimental evidence from the other typed isolates indicated that some trem families were shared while other trem families were harbored exclusively by pb01-like species or s1/ps species. it would be interesting to verify the occurrence of distinct trem families in a larger number of p. lutzii and p. brasiliensis isolates from different origins, and from different geographic regions, also including the phylogenetic species ps not represented here, in such a way as to expand and explore the potential of these newly presented genetic agents as sources of phylogenetic and epidemiological knowledge. 
although tremd and treme share about 70% similarity, they are specific to p. brasiliensis and p. lutzii, respectively. elements from each family have 65-80% sequence similarity with other tc1/mariner elements. the results may contribute to our understanding of the organization and architecture of genomes in the genus paracoccidioides. in this study the identification and characterization of class ii transposable elements in the genomes of these fungi was carried out. a genomic survey for dna transposons in the sequence assemblies of paracoccidioides, a genus recently proposed to encompass species p. brasiliensis and p. lutzii, has been completed. tremc and h elements would have been present in a hypothetical ancestor common to p. brasiliensis and p. lutzii, while trema, b and f elements were either acquired by p. brasiliensis or lost by p. lutzii after speciation. new families of tc1/mariner dna transposons in the genomic assemblies of the paracoccidioides species complex are described. the distribution of trem elements varied between the genomic sequences characterized as belonging to p. brasiliensis and p. lutzii. this suggests that these elements could either have been present in a hypothetical common ancestor and have evolved divergently after the split between p. brasiliensis and p. lutzii, or have been independently acquired by horizontal transfer. the expansion of trem in a putative ancestor common to the species p. brasiliensis and p. lutzii would have given origin to tremc and tremh, while other elements could have been acquired or lost after speciation had occurred. paracoccidioides brasiliensis is a thermodimorphic fungus, the etiological agent of paracoccidioidomycosis, the most important systemic mycoses in latin america.
 paracoccidioides brasiliensis is a thermodimorphic fungus, the etiological agent of paracoccidioidomycosis, the most important systemic mycoses in latin america. three isolates corresponding to distinct phylogenetic lineages of the paracoccidioides species complex had their genomes sequenced. in this study the identification and characterization of class ii transposable elements in the genomes of these fungi was carried out.  a genomic survey for dna transposons in the sequence assemblies of paracoccidioides, a genus recently proposed to encompass species p. brasiliensis and p. lutzii, has been completed. eight new tc1/mariner families, referred to as trem, labeled a through h were identified. elements from each family have 65-80% sequence similarity with other tc1/mariner elements. they are flanked by 2-bp ta target site duplications and different termini. encoded ddd-transposases, some of which have complete orfs, indicated that they could be functionally active. the distribution of trem elements varied between the genomic sequences characterized as belonging to p. brasiliensis and p. lutzii. tremc and h elements would have been present in a hypothetical ancestor common to p. brasiliensis and p. lutzii, while trema, b and f elements were either acquired by p. brasiliensis or lost by p. lutzii after speciation. although tremd and treme share about 70% similarity, they are specific to p. brasiliensis and p. lutzii, respectively. this suggests that these elements could either have been present in a hypothetical common ancestor and have evolved divergently after the split between p. brasiliensis and p. lutzii, or have been independently acquired by horizontal transfer.  new families of tc1/mariner dna transposons in the genomic assemblies of the paracoccidioides species complex are described. families were distinguished based on significant blast identities between transposases and/or tirs. the expansion of trem in a putative ancestor common to the species p. brasiliensis and p. lutzii would have given origin to tremc and tremh, while other elements could have been acquired or lost after speciation had occurred. the results may contribute to our understanding of the organization and architecture of genomes in the genus paracoccidioides. 