Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Zisupton—A Novel Superfamily of DNA Transposable Elements Recently Active in Fish Astrid Böhne,1 Qingchun Zhou,2 Amandine Darras,1 Cornelia Schmidt,2 Manfred Schartl,2 Delphine Galiana-Arnoux,1 and Jean-Nicolas Volff*,1 1 Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Ecole Normale Supérieure de Lyon, Lyon, France 2 Department of Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg, Germany *Corresponding author: E-mail: [email protected]. Associate editor: Norihiro Okada Abstract Key words: transposon, platyfish, sex chromosomes, SUMO, Ulp1 protease, HMGXB3, testis. Introduction Transposable elements (TEs) are mobile genetic entities found in the genome of almost all organisms. They are extremely abundant in many genomes, reaching over 90% of the DNA content in some plants (Bennetzen 2000; Jurka et al. 2007). TEs are traditionally classified into two large classes according to their transposition mechanism (Wicker et al. 2007; Kapitonov and Jurka 2008). Both TE classes include autonomous and nonautonomous sequences. Autonomous elements encode proteins necessary for their own transposition. In contrast, nonautonomous elements do not carry the complete set of genes required for transposition and use proteins encoded by autonomous elements for their mobilization. Class I elements, also called retroelements, spread throughout genomes via retrotransposition. This process is based on the reverse transcription of a TE-encoded RNA into cDNA and the integration of the new copy into another site of the genome. This ‘‘copy and paste’’ mechanism leads to an increase in copy number of the retroelement. The second class of mobile sequences is constituted by DNA transposons (class II elements). In contrast to retroelements, most class II elements transpose via a ‘‘cut and paste’’ mechanism. Transposition is catalyzed by an enzyme called transposase, which is responsible for the excision of the element from one site and its integration into another site. Other class II elements, rolling-circle DNA transposons called Helitrons, have been proposed to transpose by single-strand transfer followed by replication through the cellular machinery to create the second strand (Kapitonov and Jurka 2001, 2007). Finally, TEs known as Polintons or Mavericks are thought to transpose via selfsynthesis catalyzed by a DNA polymerase encoded by the element itself (Feschotte and Pritham 2005; Kapitonov and Jurka 2006; Pritham et al. 2007). Many different genomics effects have been attributed to TEs. Beside insertions, they can generate various types of rearrangements through transposition and recombination, such as deletions, duplications, or inversions (Gray 2000). TEs can also modify the epigenetic context and therefore © The Author 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: [email protected] Mol. Biol. Evol. 29(2):631–645. 2012 doi:10.1093/molbev/msr208 Advance Access publication August 27, 2011 631 Research article Transposable elements are widespread mobile DNA sequences able to integrate into new locations within genomes. Through transposition and recombination, they significantly contribute to genome plasticity and evolution. They can also regulate gene expression and provide regulatory and coding sequences (CDSs) for the evolution of novel gene functions. We have identified a new superfamily of DNA transposon on the Y chromosome of the platyfish Xiphophorus maculatus. This element is 11 kb in length and carries a single CDS of 24 exons. The N-terminal part of the putative protein, which is expressed in all adult tissues tested, contains several nucleic acid– and protein-binding domains and might correspond to a novel type of transposase/integrase not described so far in any transposon. In addition, a testis-specific splice isoform encodes a C-terminal Ulp1 SUMO protease domain, suggesting a function in posttranslational protein modification mediated by SUMO and/or ubiquitin small peptides. Accordingly, this element was called Zisupton, for Zinc finger SUMO protease transposon. Beside the Y-chromosomal sequence, five other very similar copies were identified in the platyfish genome. All copies are delimited by 99-bp conserved subterminal inverted repeats and flanked by copy-specific 8-nt target site duplications reflecting their integration at different positions in the genome. Zisupton elements are inserted at different genomic locations in different poeciliid species but also in different populations of X. maculatus. Such insertion polymorphisms between related species and populations indicate relatively recent transposition activity, with a high degree of nucleotide identity between species suggesting possible implication of horizontal gene transfer. Zisupton sequences were detected in other fish species, in urochordates, cephalochordates, and hemichordates as well as in more distant organisms, such as basidiomycete fungi, filamentous brown algae, and green algae. Possible examples of nuclear genes derived from Zisupton have been identified. To conclude, our analysis has uncovered a new superfamily of DNA transposons with potential roles in genome diversity and evolutionary innovation in fish and other organisms. MBE Böhne et al. · doi:10.1093/molbev/msr208 the expression pattern of host genes (Lippman et al. 2004; Haun et al. 2009). Finally, they can be considered as a kind of evolutionary toolbox for the host, which can be used as a source of new regulatory and coding sequences (CDSs) (molecular domestication, Volff 2006; Bourque 2009). Importantly, genomes can considerably differ in their TE content at both the quantitative and qualitative levels, and a same type of TE can have very different invasive success in different species (Böhne et al. 2008). Hence, TEs have the potential to generate biodiversity and evolutionary transitions through lineage-specific mutations and molecular domestication. Occasionally, TEs can be transferred between reproductively isolated species through horizontal transfer. This process, which occurs independently from classical parent-to-offspring inheritance, also plays a role in genomic diversity between species (Schaack et al. 2010). Examples of horizontal transfer of TEs in vertebrates remain rare. The numerous families of TEs identified in fish have been proposed to be involved in biodiversity and speciation in this group of animals (Volff et al. 2003; Volff 2005). With almost 28,000 described species, fish make up roughly 50% of all extant vertebrate species (Nelson 2006). They show an exceptional diversity in body shape and plan, coloration, ecology, behavior, social systems, and reproduction, among others. Sex determination is particularly variable in fish. Fish sex chromosomes are generally young and rarely heteromorphic, in contrast to differentiated and degenerated sex chromosomes in mammals and birds (Ezaz et al. 2006). TEs frequently accumulate on sex chromosomes, particularly in regions of suppressed recombination. TEs and other repeats might play a role in the differentiation process leading to the formation of heteromorphic sex chromosomes during evolution. Several new families of TEs in fish have been identified for the first time on the sex chromosomes of the platyfish Xiphophorus maculatus. This poeciliid freshwater fish belongs to a genus currently comprising 26 species inhabiting Atlantic drainages of Mexico, Guatemala, and Honduras (Kallman and Kazianis 2006). Xiphophorus has been used as a model to study melanoma formation (Gordon 1927; Kosswig 1928; Meierjohann and Schartl 2006), sexual selection (Offen et al. 2009), reproduction (Cummings and Gelineau-Kattner 2009), behavior (Fernandez et al. 2008), and sex determination and sexual development (Schultheis et al. 2006; Böhne et al. 2009; Lampert et al. 2010). Xiphophorus maculatus is particularly used to study sex chromosome structure and evolution in poeciliids, with the aim of identifying the master sex-determining gene through positional cloning. For this purpose, bacterial artificial chromosome (BAC) contigs covering the sex-determining region of the X and Y chromosomes have been constructed and partially analyzed (Froschauer et al. 2002). This region was found to be infested by TEs (Volff et al. 1999; Volff, Körting, Altschmied, et al. 2001; Volff, Körting, Froschauer, et al. 2001; Froschauer et al. 2002; Zhou et al. 2006, 2010). Here, we describe a new type of DNA transposon called Zisupton, which was identified on the Y chromosome of the platyfish. The Zisupton single coding region encodes a protein with nucleic acid–binding and protein interaction domains 632 as well as Ulp1 SUMO protease and F-box motifs specifically expressed in testis. We provide evidence for the presence of Zisupton in divergent organisms and for its recent activity and possible horizontal transfer in Xiphophorus. Materials and Methods Fish Platyfish (X. maculatus, population Rio Jamapa WLC1274, closed colony stock derived from Jp163A) were kept under standard conditions at the PRECI fish facility of the IFR128 Biosciences Gerland-Lyon Sud (Lyon, France). Samples from X. couchianus, X. xiphidium, X. variatus, X. milleri, X. clemenciae, X. hellerii, X. andersi, and X. nezahualcoyotl were obtained from the fish facility of the Biozentrum at the University of Würzburg (Germany). In Silico Sequence Analysis Zisupton-related protein and nucleotide sequences were identified using BLAST/BLAT and PSI-BLAST searches against GenBank databases (www.ncbi.nlm.nih.gov/blast/ Blast.cgi). BLAST analysis was also conducted using the current Ensembl genome assemblies of zebrafish (Danio rerio), medaka (Oryzias latipes), green spotted puffer fish (Tetraodon nigroviridis), fugu (Takifugu rubripes), three-spined stickleback (Gasterosteus aculeatus), and sea squirt (Ciona intestinalis) (www.ensembl.org; version 57, March 2010). Genome draft from acorn worm (Saccoglossus kowalevskii) (http://www.hgsc.bcm.tmc.edu/project-species-o-Acorn% 20worm.hgsc) and amphioxus (Branchiostoma floridae) (http://genome.jgi-psf.org/cgi-bin/runAlignment?db5Brafl1 &advanced51) was also analyzed. Hits were generally considered as significant below an E value cutoff of 1 1005. Automatic sequence annotation was refined manually using expressed sequence tag data and related protein sequences with the help of FGENESH on the softberry web server (http://linux1.softberry.com). Nucleotide and protein sequences were loaded into BioEdit (Hall 1999) and aligned using MUSCLE (Edgar 2004a, 2004b), T-Coffee (Notredame et al. 2000), and COBALT (Papadopoulos and Agarwala 2007). Alignments were checked manually and ambiguously aligned regions were removed before phylogenetic analysis. The phylogenetic tree was constructed on a 166 amino acid alignment generated through concatenation of domains conserved in divergent Zisupton proteins. Maximum likelihood reconstruction was done using PhyML 3.0 (Guindon and Gascuel 2003; Guindon et al. 2010) with 100 bootstraps under the LG model. Similar results were obtained using the neighbor joining method as implemented in MEGA4 (Tamura et al. 2007). MEGA4 was also used to visualize phylogenetic trees. Syntenic relationships between genomes were established using Genomicus (Muffato et al. 2010). Conserved protein regions in divergent Zisupton proteins were identified using MEME (http://meme.nbcr.net/ meme4_6_1/cgi-bin/meme.cgi). Annotation of conserved protein motifs was performed with MotifScan (http:// myhits.isb-sib.ch/cgi-bin/motif_scan) and NCBI Conserved Domain Search (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi). MBE Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208 Ka/Ks calculation for puffer fish Zisupton-related sequences was performed using Ka/Ks Calculator under the Modified version of Yang-Nielsen method model (Zhang et al. 2006) on a pairwise nucleotide alignment done with MUSCLE (Edgar 2004a, 2004b). Fisher’s exact test and chi square with Yates’ correction were performed on http://www.graphpad.com/quickcalcs/contingency1.cfm. Xiphophorus maculatus Zisupton sequence has been deposited in GenBank under accession number JN255571. Molecular Cloning DNA fragments were cloned into pBluescript II KSþ (Stratagene) or using the TOPO TA Cloning Kit for Sequencing (Invitrogen). BAC digestions for shotgun subcloning were performed on 500 ng DNA under conditions recommended by the manufacturer (Fermentas), with EcoRI (unique restriction site in exon 3 of Zisupton) and MscI (unique restriction site in the 3# noncoding region of Zisupton). Cloning into pBluescript II KSþ was done using Klenow fragment, T4 polynucleotide kinase, shrimp alkaline phosphatase, and T4 DNA ligase (all from Fermentas). Ligations were done overnight at 4 °C and subsequently used to transform chemical competent DH5a bacteria according to Ausubel et al. (2010). Miniplasmid preparations were done from 5-ml overnight cultures using either standard protocols or the QIAprep Spin Miniprep Kit (Qiagen). DNA sequencing was carried out by GATC Biotech (Constance, Germany). Polymerase Chain Reaction Amplification Polymerase chain reactions (PCRs) were performed using the Taq DNA Polymerase Recombinant (Invitrogen), with primer-dependent annealing temperatures ranging from 55 to 65 °C. Single-primer PCRs to characterize Zisupton insertions were performed using one specific internal Zisupton primer (5#-CCTTTGCTCATACAGTGCGG-3#, final concentration 6 ng/ll) on 100 ng of genomic DNA (adapted from Parks et al. 1991). After an initial incubation step at 94 °C for 5 min, the thermal cycling program was as follows: 1) 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min during 20 cycles; 2) 94 °C for 30 s, 40 °C for 30 s, and 72 °C for 2 min during 30 cycles; and 3) 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min during 30 cycles. Amplification of the X. hellerii-specific insertion was done using the QIAGEN Long Range PCR kit with primers (5#-TCACATTTCAGCCACTATTTG-3#) and (5#-CTGTCGGAATCAGGCAAGT3#). Primer sequences for amplification and sequencing are available upon request. Primers were ordered from Invitrogen. Expression Analysis Total RNA was extracted from male and female tissues (brain, eye, gill, liver, muscle, ovary, skin, spleen, and testis) and embryos using the TRI Reagent (Molecular Research Center, Inc). After DNase treatment (Fermentas), reverse transcription was performed on 1 lg of total RNA using the RevertAid First Strand Synthesis kit (Fermentas) and random hexamer primers. Classical real-time (RT) PCR experiments for Zisupton (fig. 1) were performed with primers (5#-CTATCAATGGCACTGGCTT-3#) and (5#-TGGCATTCAGGGCAGTGT CC-3#) for the testis-specific region and (5#-GCAGTGGG ACAGACACAACTT-3#) and (5#-CAGCAAGTGATCGTTGAAG-3#) for the ubiquitously expressed region. PCR was done with an annealing temperature of 58 °C (35 cycles). The amount of cDNA template was adjusted between reactions using a beta-actin gene control. Quantitative RT-PCRs were done on 2 ll of serial dilutions of cDNA starting from a dilution of factor 20 using IQTM Custom SYBR Green Supermix (Bio-Rad). PCR amplification was monitored with a CFX96 Real-time System (Bio-Rad). After one incubation step at 95 °C for 3 min, the thermal cycling protocol was as follows: 95 °C for 10 s, 55– 62 °C (primer-dependent annealing temperature) for 20 s, and 72 °C for 15 s during 40 cycles. Primers were designed manually and ordered from Invitrogen. For each primer set, the efficiency of the PCR reaction was measured in duplicate on serial dilutions (factor 10) of the cDNA sample. RT-PCR efficiencies (E) were calculated for each reaction from the slope of the standard curve using the equation E 5 10(1/slope) 1, as implemented in the software BioRad CFX manager V1.5, with 100% efficiency as an indicator of a robust assay. All results reported are average of at least two independent reverse transcription reactions and two RT-PCR experiments for each reaction. For quantification, data were analyzed using the 2(-Delta Delta C(T)) method (Livak and Schmittgen 2001), with normalization to the housekeeping gene ef1a (elongation factor 1 alpha). Expression was normalizedto eyetissue.Primer efficiencyandspecificitywas testedon genomic DNA from each species before RT-PCR experiments. Results Zisupton Is Present on the Y But Not on the X Chromosome in the Sex-Determining Region of the Platyfish X. maculatus In order to identify DNA sequences differentiating the Y from the X chromosome of X. maculatus in the sexdetermining region, BAC contigs from each type of sex chromosome were assembled and compared (Froschauer et al. 2002). Sequence comparison revealed an approximately 11 kb sequence present in two overlapping Y-linked BAC clones (F1 and O4) but absent from the homologous X-linked clone K26. This sequence, which was called Zisupton, was found to contain one unique CDS (see below) (fig. 1). Through exon walking on cDNA and rapid amplification of cDNA ends-PCR on testis RNA/cDNA, the exon–intron structure of Zisupton was characterized. The putative full-length CDS (from ATG to stop codon) was 4,182 bp in length and consisted of 24 exons. Zisupton Encodes a Putative Nucleic Acid–Binding Protein Involved in Posttranslational Protein Modification The predicted full-length protein (1,393 amino acids) of the Zisupton element contained putative nucleic acid–binding 633 Böhne et al. · doi:10.1093/molbev/msr208 MBE FIG. 1. Gene structure, predicted protein domains and expression of Zisupton in the platyfish. (A) The Y-specific copy of Zisupton identified in BAC clones F1/O4 (24 exons). Symbols in the schematic protein above the gene structure of Zisupton indicate the conserved motifs. Numbered boxes are exons, black boxes are introns, and untranslated regions are in gray. (B) RT-PCR analysis of the expression of Zisupton. Black arrows indicate primer positions. The larger fragment in the RT-PCR experiment for the ubiquitous transcript corresponds to an unspliced form, as demonstrated by DNA sequencing of RT-PCR products. and protein interaction domains in its N-terminal part: two C2H2 zinc fingers, a C3H zinc finger called the SWIM (SWI2/SNF2 and MuDR) domain, and a SAP (SAF-A/B, Acinus, and PIAS) domain (fig. 1). C2H2 zinc fingers are often a characteristic attribute of transcription factors with sequence-specific DNA binding (for review, see Rosenfeld and Margalit 1993; Iuchi 2001). C2H2 zinc fingers can also confer binding to RNA (Brown 2005) or proteins (Brayer and Segal 2008). The function of SWIM domains, characterized as cysteine–histidine zinc-chelating domains, remains unclear. However, due to structural similarities, properties related to those of C2H2 zinc fingers have been proposed (Makarova et al. 2002). Interactions of SWIM domains with either DNA or RNA have been reported for some proteins (Banerjee et al. 2004; Cai et al. 2008; Lin et al. 2008). The SAP motif is a DNA-binding domain found in nuclear proteins (Aravind and Koonin 2000; Suzuki et al. 2009). This motif may bind to specific DNA sequences or structures and possibly acts on chromosomal organization (Aravind and Koonin 2000). Hence, the Zisupton protein might be able to bind to DNA or RNA. A repetitive motif of 14 amino acids (PAATDIQATQAPVR) of unknown function was located between both C2H2 fingers. The C-terminal part of the Zisupton protein contained an Ulp1 (ubiquitin-like protease 1) domain. This domain is 634 a SUMO (Small Ubiquitin-like MOdifier)-specific protease belonging to the subfamily of C48 cysteine peptidases (Barrett and Rawlings 2001). SUMO proteases are involved in the cleavage of SUMO from SUMO-conjugated substrates or poly-SUMO chains and the maturation of SUMO itself (for review, see Yeh 2009). The Ulp1 protease domain of Zisupton was flanked on its C-terminal side by a putative F-box motif. This domain implicated in protein–protein interactions is present in F-box proteins that act as adapter components of SCF (Skp, Cullin, F-box containing) complexes. SCF complexes are a family of huge modular E3 ubiquitin ligases (Craig and Tyers 1999; Jackson and Eldridge 2002). Taken together, these observations suggested that the full-length Zisupton protein might be involved in posttranslational protein regulation mediated by SUMO and/or ubiquitin small peptides. Interestingly enough, the SWIM domain of the MEX E3 ubiquitin ligase is involved in ubiquitination (Nishito et al. 2006), suggesting that this domain of Zisupton might also play a role in posttranslational protein modification. Zisupton Full-Length Isoform Is Testis Specific Through exon walking on testis cDNA and expression analysis on adult organs and tissues, we could identify two splice variants of Zisupton. One of them, encoding a shorter Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208 isoform, was lacking exon 15 and contained a premature stop codon at the beginning of exon 16 (fig. 1B). At the protein level, only the N-terminal part with both C2H2 zinc fingers, the SWIM zinc finger and the SAP domain were present. By RT-PCR, expression was detected in all adult organs and tissues tested (brain, eye, gill, liver, muscle, ovary, skin, spleen, and testis). The additional upper band in figure 1B corresponded to a nonspliced product as confirmed by sequencing of the PCR products. In contrast, the longer splice variant, in which inclusion of exon 15 restored the complete open reading frame (ORF), was found to be expressed only in testis. Hence, the Zisupton isoform with Ulp-1 and F-box domains was apparently testis specific in adults. Zisupton Is Multicopy in Platyfish Genome In order to test for the presence of additional copies of Zisupton in the genome of X. maculatus, genomic DNA of females and males was cut with EcoRI (one unique restriction site in Zisupton), and Southern blot DNA hybridization was performed with a Zisupton-specific probe derived from exon 3. In males, several signals were obtained in addition to the Y-specific copy, indicating that Zisupton was multicopy (data not shown). All these additional signals were also found in females, showing that Zisupton is not Y specific. A BAC genomic library of male platyfish DNA (Froschauer et al. 2002) was screened by Southern blot DNA hybridization with the same Zisupton probe. Sixty-four Zisupton-containing clones were isolated and analyzed through restriction fragment length polymorphism (RFLP) analysis. Besides BAC clones containing the Y-specific copy already characterized, five further RFLP groups were identified (fig. 2). None of them belonged to the BAC contigs covering the sex-determining region of the X and Y chromosomes of the platyfish. The Zisupton-positive fragments from these groups were found in both males and females (data not shown). Hence, these copies were located either on autosomes or in the pseudoautosomal regions of the sex chromosomes. To conclude, at least five copies were present in platyfish females and at least six copies in males. Zisupton Is a DNA Transposon For each RFLP group, Zisupton and its flanking regions were sequenced directly from representative BAC clones (fig. 2). All six copies showed a high level of nucleotide identity (.99%) in exons and introns. Strikingly, sequence identity between copies always stopped at the same precise base pair position, giving sharp 5# and 3# boundaries. No sequence similarity was found between flanking sequences from different groups, indicating that they indeed corresponded to different copies of Zisupton. Comparison of 5# and 3# extremities revealed the presence of 99 bp terminal inverted repeats (TIRs) conserved in all Zisupton copies. These repeats were subterminal, the ends of the element being constituted by 8-nt segments with no sequence similarity between both ends. Target site duplications (TSDs) of 8 bp flanked each copy of Zisupton. TSDs from different copies were different and did not display any obvious common motif. TIRs and TSDs are structural hallmarks of TE insertions. Therefore, Zisupton might MBE correspond to a DNA TE (10,784 bp in length). The absence of similarity between TSDs from different insertions indicated that Zisupton integration is not site specific. Strikingly, three copies (copies III, IV, and V), clearly corresponding to different insertions, were 100% identical over their complete length (almost 11 kb) (fig. 2). They differed from the Y-specific copy at only two nucleotide positions: an A-G transition in exon 8, leading to a histidine–arginine nonconservative change (CAT-CGT) outside of the conserved domains identified in Zisupton, and a T missing in the 3# untranslated region of the Y copy. Neither frameshift nor nonsense mutations were found in these four copies. Taken together, the presence of (almost) identical noncorrupted copies suggested that Zisupton was recently—and might be still—active in the genome of the platyfish. The two remaining insertions have been inactivated through mutations. Sequencing of regions surrounding the different Zisupton insertions revealed that at least two copies were inserted in repeat-containing regions (copies I and IV). Copy III was inserted directly 3# behind the pol ORF of a Bel/Pao long terminal repeat retrotransposon. Copy IV insertion was located in an intergenic region 360 nt 5# from an apparently intact glucosidase gene (1.3 kb). The Y-chromosomal copy was found to be inserted in a nonautonomous Zisupton-like element called little-Zisupton (liZi, 3,372 bp). LiZi was located at the same position on the X and on the Y chromosome. LiZi was delimited by TIRs (21 nt) and flanked by TSDs (10 nt), indicating that it also corresponded to a TE. Detailed examination of the insertion on the Y chromosome indicated that Zisupton TSD sequence corresponded to the first 5# nucleotides of the 3# TIR of liZi. Hence, we assume that Zisupton was integrated into the 3# TIR of liZi on the Y chromosome. Recent Transposition Activity and Putative Horizontal Transfer of Zisupton in the Poeciliidae Fish Family Using Southern blot hybridization and PCR amplification with X. maculatus primers, we could detect Zisupton elements in other fish species from the family Poeciliidae (interspecific divergence 3–20 My; Hrbek et al. 2007). We amplified by PCR, sequenced and assembled large overlapping fragments of Zisupton from other Xiphophorus species including X. clemenciae, X. andersi, X. milleri, X. nezahualcoyotl, X. xiphidium, and X. hellerii (table 1). Strikingly, all Zisupton sequences from these species were extremely similar, with at least 99.9% of nucleotide identity. Inclusion of different PCR controls and detection of rare species-specific single nucleotide polymorphisms indicated that these results were not due to contaminations. No rearrangements such as deletions, insertions, or inversions were detected in the amplified fragments. In order to test if the low interspecific variability observed for Xiphophorus was in the range of variation observed for other nuclear genes, we amplified and sequenced parts of the b-actin genes from different species. Published data for exon 3 of rag1 were also included (Meyer et al. 2006). Only presumed neutral changes (substitutions at synonymous sites 635 Böhne et al. · doi:10.1093/molbev/msr208 MBE FIG. 2. Structural comparison of different Zisupton insertions in Xiphophorus maculatus. Boxes at the boundaries of the sequences: target site duplications (TSDs), white arrowheads: terminal inverted repeats (TIRs), white boxes: Zisupton exons, black boxes: introns, little white boxes above exon 3: repetitive sequence region showing numbers of repetitions, black letters: synonymous/silent nucleotide substitutions, and gray letters in italics: nonsynonymous nucleotide changes. Neighbor sequences were identified using BLAST and are marked as boxes; gene names are given according to most significant BLAST hits. Dashed line indicates incomplete sequence data for copy V. in exons and substitutions in intronic sequences) were considered. No intronic data were available for rag1. In most cases, Zisupton transposons from two Xiphophorus species were significantly more similar than nuclear genes (table 1). This was particularly true in comparisons with X. nezahualcoyotl, which was the most divergent species included compared with X. maculatus. As expected, differences were less or not significant for species closely related to X. maculatus, such as X. milleri and X. xiphidium. Even if these data must be completed by other nuclear sequences for extensive comparison, they are consistent with horizontal transfer of Zisupton in Xiphophorus. In order to investigate the transposition dynamics of Zisupton, we tested X. maculatus insertion sites by PCR on genomic DNA of different poeciliid species as well as on different populations of X. maculatus. Primers were designed for 5# and 3# flanking genomic regions and used each in combination with a Zisupton-specific internal primer to test for the presence of the insertion. Primers for 5# and 3# flanking genomic regions were combined to test for the presence of ‘‘empty’’ sites without transposon insertion. We performed this test for the six Zisupton insertions identified in X. maculatus. For all 10 Xiphophorus species tested, the flanking regions were conserved enough to be amplified by PCR. For copy V, only the 3# insertion test could be done since no sequence information was available for the 5# flanking genomic region. Results demonstrated that all six genomic sites carrying a Zisupton insertion in X. maculatus were devoid of insertions in other Xiphophorus species. Similarly, no orthologous insertion was detected in non-Xiphophorus species (Gambusia affinis, several Poecilia species). We also tested two X. maculatus populations (Rio Usumacinta and South 636 Rio Grijalva) different from the population used to construct the genomic BAC library (Rio Jamapa). Both Rio Usumacinta and Rio Grijalva populations contained Zisupton, as shown by PCR and Southern blot hybridization (data not shown). Strikingly, all the above characterized insertions were Rio Jamapa specific; only empty sites were detected in the Rio Usumacinta and Rio Grijalva population. None of the empty sites contained any ‘‘footprint,’’ that is, any small rearrangement left after transposon excision. These findings indicated recent activity of the transposon after separation of the different populations. We were able to identify a further Zisupton insertion in the swordtail X. hellerii. Using only one PCR primer matching the second exon of Zisupton, the 5# flanking region of an X. hellerii insertion was amplified by single-primer PCR. The fragment obtained was cloned and sequenced. The flanking region, showing similarity with the hAT transposon Trilian from the puffer fish T. rubripes (Smit 2002), did not correspond to any insertion site identified in X. maculatus. PCR assays showed that this insertion was not present in other Xiphophorus species. Importantly, the sequence of Zisupton in this X. hellerii-specific insertion is identical over 4 kb with the 5# part of the Y copy of Zisupton in X. maculatus, excluding PCR contamination and suggesting again interspecific horizontal transfer. We also tested for the presence of intrapopulational insertion polymorphism in the Rio Jamapa strain of X. maculatus using 24 males and 17 females. The Y-specific insertion was present in all males and absent from all females tested. No insertion polymorphism was detected for copies I to IV. In contrast, copy V was apparently absent from 10 males and 6 females, as tested by ‘‘3# insertion’’ PCR. However, absence of sequence data for the 5# region MBE Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208 C2H2 repetitive motif SWIM C2H2 Ulp1 protease SAP STOP ATG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Maculatus 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 11.00 10.00 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 B E G L O/T S M Embryos head tail 15.00 10.00 3.00 2.00 5.00 1.00 0.00 0.00 O/T S M B E G L O/T S M L 5.00 4.00 3.00 2.00 1.00 0.00 S B M S M O6days E Clemenciae G L OandE6days O/T S head tail 500.00 400.00 300.00 200.00 100.00 0.00 B E G L O/T S B M E G L O/T S M Couchianus 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 B M Embryos Millerii 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 6.00 O/T O/T Variatus 7.00 L 0.00 G Couchianus Variatus 16.00 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 G 0.50 E 160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00 4.00 E 1.00 Xiphidium 5.00 B 1.50 B 6.00 L 2.00 0.00 OandE6days 7.00 G 2.50 300.00 Millerii 20.00 E 400.00 100.00 Xiph idium B 3.00 200.00 O6days 25.00 Maculatus Embryonic 500.00 E G L O/T S M B E G B E G L O/T S M Nezahualcoyotl 7.00 12.00 6.00 8.00 4.00 3.00 6.00 2.00 4.00 1.00 2.00 0.00 0.00 B E G L O/T S M B E G Andersi L T/O S M 100.00 0.00 B E G S M O/T S M 0.00 0.00 B E G L T S M S T S M 8.00 6.00 4.00 400.00 200.00 O/T 10.00 600.00 1.00 L Hellerii 800.00 2.00 O/T L 1000.00 3.00 L 200.00 1200.00 4.00 G 300.00 Andersi 5.00 E 400.00 Hellerii 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 B 500.00 400.00 350.00 300.00 250.00 200.00 150.00 100.00 50.00 0.00 10.00 5.00 2.00 0.00 B E G L O/T S M B E G L M FIG. 3. Quantitative RT-PCR expression analysis of Zisupton in the genus Xiphophorus. The expression of Zisupton was tested using two primer combinations, one reaching from exon 3 to exon 4 (left side, gray box) and the other one from exon 15 to exon 19 (right side, black box). Amplified fragments are marked with gray and black boxes, respectively. B, brain; E, eye; G, gill; L, liver; O, ovary; T, testis; S, skin; M, muscle; O6d, ovary 6 days after fertilization; OþEmb, Ovary with embryos 10 days after fertilization; Em, embryos 10 days after fertilization; H, head of 1-day-old fry; and Bo, body of 1-day-old fry. Male tissues are in black, female tissues in white, and embryonic samples in gray. flanking this insertion did not allow to confirm these results through ‘‘5# insertion’’ and ‘‘empty site’’ PCR tests. Taken together, insertion polymorphism analysis in Xiphophorus, added to the presence of noncorrupted (almost) identical copies of Zisupton, suggests that this transposon was recently active in poeciliids and could have been subject to horizontal transfer. Expression Analysis of Zisupton in the Genus Xiphophorus First classical RT-PCR experiments (fig. 1) showed that exon 15 of Zisupton is only expressed in testis in X. maculatus. Without this exon, the Zisupton-transcript contains a premature stop codon in exon 16. In testis, inclusion of exon 15 restores the ORF and leads to the production of a longer protein. In order to know if this splicing and expression pattern was conserved among Xiphophorus species, we tested several species in quantitative RT-PCR experiments (fig. 3): X. maculatus Rio Jamapa as a reference, X. xiphidium, X. milleri, X. variatus, X. couchianus, X. andersi as other platyfishes and the swordtails X. clemenciae, X. nezahualcoyotl, and X. hellerii. The amplified fragments and primer-binding sites were 100% identical between these species, allowing direct interspecific comparison of expression levels. We tested two primer combinations for Zisupton: the first combination was located in the 5# part of 637 MBE Böhne et al. · doi:10.1093/molbev/msr208 Table 1. Comparison of interspecific sequence divergence for Zisupton and two nuclear genes, b-actin and rag1. Sequence Zisupton b-actin rag1 X. X. X. X. X. X. Species clemenciae andersi milleri nezahualcoyotl xiphidium hellerii Sequence length in nt 2785 2785 2785 2785 2785 2785 Synonymous changes in exons/ introns compared with X. maculatus 0/0 0/0 0/0 0/1 0/3 0/3 X. clemenciae 584 2/6 X. andersi 821 0/2 X. milleri 821 1/2 X. nezahualcoyotl 821 2/7 X. xiphidium 821 0/2 X. hellerii 821 0/4 X. clemenciae 1,573 9/— X. andersi 1,573 4/— X. milleri 1,573 1/— X. nezahualcoyotl 1,573 7/— X. xiphidium 1,573 4/— X. hellerii 1,573 8/— P-value <0.0001** <0.0001** 0.052 0.078 0.012* 0.012* <0.0001** <0.0001** 0.312 0.699 0.051 0.085 0.0001** 0.0003** 0.017* 0.003* 0.36 0.77 0.004** 0.008** 0.261 0.443 0.022* 0.026* NOTE.—Sequences were compared with X. maculatus (for Zisupton, the Y-specific sequence was used as a reference). Nucleotide divergence was calculated using only substitutions at synonymous sites in exons (for all three sequences, first value) and substitutions in intronic sequences (for Zisupton and b-actin, second value). Two-tailed P values (first values: Fisher’s exact test; second values: chi square with Yates’ correction) are provided to assess if the interspecific divergence observed between nuclear genes is statistically higher than the divergence observed between Zisupton elements (calculated using the sum of synonymous changes; **extremely/very statistically significant, *statistically significant). Zisupton and b-actin data have been obtained by PCR; data for exon 3 of rag1 are from Meyer et al. (2006). No intronic sequence was available for rag1. Zisupton (exon 3 to exon 4, gray box, fig. 3) and the second one started from the exon specifically expressed in X. maculatus testis (exon 15) until exon 19 (black box, fig. 3). The observed expression pattern with the first primer combination located in the 5# part confirmed a broad expression pattern in adult tissue. However, expression was stronger in testis and male/female brain in all tested species. This part was also expressed in head of 1-day-old fry of X. maculatus. With the primer combination amplifying exons 15–19, exclusive testis-specific expression reaching much higher expression levels was observed in X. maculatus, X. xiphidium, X. milleri, X. clemenciae, and X. nezahualcoyotl. Xiphophorus andersi showed additionally high expression in male brain. Xiphophorus hellerii displayed lower expression than the other species tested, with an expression pattern more similar to that obtained with the first primer combination. Xiphophorus variatus and X. couchianus, two platyfishes, did not show any preferential testis expression with the second primer combination, with overall lower gene expression. All together, the 3# part is, if strongly expressed, restricted to testis, with preferential expression in brain in some species. 638 Distribution of Zisupton-Like Transposons To test if Zisupton is related to any known TE in fish and other organisms, we used the platyfish Zisupton sequence to search against available TE databases (Jurka et al. 2005; Kohany et al. 2006 and http://www.repeatmasker.org). Zisupton did not match any entry and was not recognized as a TE. To gain insights into the distribution and evolution of Zisupton transposons, available sequence databases were screened in silico with Zisupton protein sequences as queries. Zisupton-related sequences were identified in all five sequenced fish genomes (zebrafish, medaka, stickleback, and the two puffer fishes T. nigroviridis and T. rubripes) (figs. 4–6). The largest number of Zisupton-related sequences (about 30 copies) was found in zebrafish. In all these species, sequences with TIRs and TSDs were identified, suggesting that Zisupton is also a transposon in other fish species. As observed in the platyfish, Zisupton inverted repeats are subterminal in other fish species. No Zisupton transposon was detected in tetrapods, suggesting loss from this lineage during evolution. Sequences were found in Ciona (urochordate) as well as in the amphioxus B. floridae (cephalochordate) and the acorn worm MBE Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208 S. kowalevskii (hemichordate). In Ciona savignyi, two very related Zisupton sequences (8.3–8.4 kb in length) were found to be integrated at different positions in the genome (accessions AACT01001128 and AACT01065526). Presence of empty sites without insertion in the genome database allowed identifying 8-nt TSDs for both insertions. Imperfect subterminal inverted repeats were identified for both sequences. Hence, Zisupton probably behaves as a TE in Ciona too. In the Zisupton proteins, C2H2 and SWIM zinc fingers were detected in fish, urochordates, cephalochordates, and hemichordates (fig. 5). The SAP motif was predicted in fish and acorn worm, the Ulp1 protease, and F-box domains only in fish. Interestingly, the amphioxus and acorn worm sequences contained protease domains like Zisupton, but these domains were different. The protease domain in amphioxus was a C19 protease, also known as ubiquitin carboxyl-terminal hydrolase. The protease in the acorn worm sequence was an OTU domain, also a cysteine protease (Makarova et al. 2000). Through PSI-BLAST analysis, divergent Zisupton-related sequences were identified in basidiomycete fungi (Lacaria, Coprinopsis, Serpula, Postia, Moniliophthora, and Cryptococcus), filamentous brown algae (Ectocarpus), and green algae (Volvox and Chlamydomonas) (figs. 4–6). Most of these sequences were multicopy and showed structures typical of TEs. In some cases, transposon ends and TSDs were precisely characterized through comparison with empty sites present in the databases. For instance, in the fungus Lacaria bicolor, two insertions of 12.8 and 13.3 kb displayed imperfect TIRs and 8-nt TSDs (accessions ABFE01001864 and ABFE01001823).In Postia placenta,another fungus, a 6-kb sequence with 277-nt TIRs and 8-nt TSDs has been identified (accession ABWF01004332). In the filamentous brown alga Ectocarpus siliculosus, a 14.7 kb Zisupton-like transposon with subterminal TIRs and 8-nt TSDs was found in the genome (accession CABU01009984). Importantly, none of the sequences recovered through PSI-BLAST did correspond to any known TE. Only the SWIM domain was detected in Zisupton proteins from fungi and green algae but not from Ectocarpus (fig. 4 and 5). Sequence alignment of predicted Zisupton proteins from divergent organisms allowed identifying four domains of higher sequence conservation containing amino acid residues potentially critical for transposition activity. Domain I contained the SWIM domain. Domains II and III (between SWIM and SAP domains in fish proteins) to IV (between SAP and Ulp1 protease domains) did not carry any amino acid signatures found in other transposon proteins. From the phylogenetic point of view, sequences from chordates/ hemichordates, fungi, green algae, and brown algae were well differentiated (fig. 6). The presence of one fish sequence in the Ciona group might be explained by in silico contamination, presence of several ancient Zisupton lineages in chordates or horizontal transfer. Domesticated Zisupton Transposons in Vertebrate Genomes In puffer fishes, only one Zisupton-related sequence was found in T. nigroviridis and two in the Fugu T. rubripes. Interestingly, synteny analysis revealed that the unique Tetraodon sequence and one Fugu sequence were orthologous and inserted at the same position in their respective genome. This indicated conservation of an insertion event that must have taken place before the split of these two species approximately 18–30 Ma (Van de Peer 2004). Neither TIRs nor TSDs were detected, questioning the functionality of both sequences as transposons. Both orthologous sequences contained an intact coding region that encoded a putative protein with similarities only to the N-terminal part of the Zisupton protein (fig. 5). When we compared the rate of nonsynonymous (Ka) with the rate of synonymous substitutions (Ks) between both puffer fish sequences, we obtained a Ka/Ks value of 0.28. This suggested that they evolved under purifying selection and probably did not correspond to pseudogenes. Hence, these sequences might correspond to a ‘‘domesticated’’ form of Zisupton performing functions useful to the host, as reported for other TEs (for review, see Volff 2006). A family of vertebrate proteins called HMGXB3 (high mobility group [HMG] box domain containing 3, also known as SMF) was found to be related to Zisupton proteins (E value 5 2 1021 for BLAST comparison between platyfish Zisupton and mouse HMGXB3; figs. 4–6). HMGXB3 proteins possess an HMG DNA-binding motif found in a variety of transcription factors that preferentially bind to distorted DNA (Thomas 2001). Significant similarity with Zisupton proteins was found in conserved domains II, II, and IV. HMGXB3 proteins lack the SWIM, SAP, ULP1 protease, and F-box domains. Presence of HMGXB3 in fish, amphibians, birds, and mammals suggested an ancient event of molecular domestication that occurred at least 450 Ma in a common ancestor of fish and tetrapods. Discussion We have described here Zisupton, a fish TE encoding a protein with DNA-binding domains and SUMO protease/ F-box motifs. Related elements were identified in urochordates, cephalochordates, hemichordates, basidiomycete fungi, filamentous brown algae, and green algae. Zisupton generally contains introns and does not encode any reverse transcriptase or other proteins typical of retroelements. Zisupton does not share any homology with already described TEs at both the nucleotide and protein levels. We therefore propose that Zisupton represents a new superfamily of DNA transposons. A region containing four conserved domains, including the SWIM motif, might correspond to a new type of transposase, which remains to be analyzed at the functional level. No aspartate/aspartate/ glutamate catalytic triad classically found in transposases and integrases could be unambiguously identified through comparison of divergent Zisupton proteins. Some of the predicted functional domains detected in Zisupton proteins have been found in other transposons. Ulp1 protease domains are encoded by GIN/Ginger transposons in animals (Bao et al. 2010; Marı́n 2010) and a large family of Mutator-like TEs in plants (Hoen et al. 2006; van Leeuwen et al. 2007). A SWIM domain has been identified in plant MuDr transposases (Makarova et al. 2002). At the structural level, subterminal inverted repeats found in fish 639 MBE Böhne et al. · doi:10.1093/molbev/msr208 I 65-556aa 76-198aa SWIM II 36-76aa Platyfish Ciona Amphioxus Saccoglossus Lacaria Coprinopsis Serpula Postia Moniliophthora Cryptococcus Chlamydomonas Volvox Ectocarpus Hmgxb3_Mouse : : : : : : : : : : : : : : -----NTWHCACTEG-------KRSCTHKATAK-----VTLTGVVKGISTYRKTCPNCS----MVYRYQDWEDGIHNFNDHLLLSIHFC-----YLSFEG -----GTWSCQCPRASG-----TSTCLHESIAK-----YGINKIWKGTTMITKLCRSCK----CITRFQNYKSGFHNYNNQIILSICLC-----FYHFCA -----NILNCTCCKI-------KRGCIHKGISK-----VGLQHISTDIEVFVRKCPQCH----HVFRYQEYEDGLLNYNNKTFLTLQLC-----YLLFEV -----NTWHCRCCK--------KRGCGHKALAR-----VAMNGLIKGVETYCRKCSLCD----LVYRYQEHCDQLHNYDDKWIFSVRLL-----YEHFEA -----LIVSCMCPASTSRSTGLSASCFHIKFLL-----IYGLNCAWESMIEVQPCGTCA---KRNIGPDCQDLGLFNYDNDRLVTHALM-----WFAYVQ -----IHTVCTCPHGMED----EVLCIHRRFIE-----VYTLTTVYVHQIQLQRCPQCPPSRRRFIGPDPRHEGLFNYNNSVLVSHELL-----WFAYVS -----IVVVCFCLR--------GSTCFHVQFFQ-----IYTLTNAWTSVIEVQSCTSCPPHKCCFIGPDLQEHGLFNLNNWLLFTHDLL-----LFAYVQ -----LGVACTCPLFKA-----QRNCTHRRVLL-----LYGLTMRNQVSIEVYPCPSCHH-HQRFIGPDLGCEGIFNWNNLFLFTHELL-----WFAFVR -----LTVTCLCPDGDS-----FRRCVHEQFYL-----VYTLTGCISRRIELQQCSQCHQRRHCYVGPEPRDIGLFNYNNTVLFTHELM-----WFAFAT -----GEKSCDCPAYRS-----NQDCVHVLLYI-----VLHTTGVEKVRLETVRCPRPGC--GRDIGPDLASHGLVNFNNEIGFSRNVF-----FFAFLK -----PRMTCTCSSFRVVGAA-VSRCLHTAYVS-----VGPVRAHLFELYSGCSCLACVMAYDGVAD------GLFRYSRSSFFCLRHM-----WQQFEA -----GSPCCSCEIYGTT----GVSCLHLEYVH-----MGPRHAAEYMLRSRCGRSACNIVYDGFGD------GLFRHSSRSYFSLRHM-----WYKFIT -----GGTGCTSSKKGS-----SGKCVHTHIVG-----LVPGPDLVEAVLKIKGCVDCGV--EAVTGDDWQEHGYFVFGETLLVHLSHM-----YLAFEA -----KDRTEKTPKAI------EASSPHPDVPS-----LLTANRLQVVTAQVKMCLNPHC--LALHSFTDIYTGLFNVGNKLLVSLDLL-----YWAFEC Platyfish Ciona Amphioxus Saccoglossus Lacaria Coprinopsis Serpula Postia Moniliophthora Cryptococcus Chlamydomonas Volvox Ectocarpus Hmgxb3_Mouse : : : : : : : : : : : : : : LSDHDY-QYSCDSCGYHPAIVVMDL-----SGGWAVITCPCGIIYSVKFNLRAESPRD-FADLLLSWKFFPNITVYDYPRGLVSHLKKRCA----LKDFSY-DFNCLKCGHSPKILIADG-----GGGVLHFRCPHGICYYFKTLLRQESARD-FVDGLLSLKQQPIVFISDVASQVAHHGENRKT----LSGHQY-EYYCIRCGHHPPVLIMDL-----SGGLLVAACPHRVVYAAKFLLRGESPRD-FVDILLSMQWQPTVTIADMPQIIAAHGNGRCP----LTDHDY-TFNCVICGSNPPVIITDL-----SGGWLSASCPHRIVYAVKFLIRAESPRD-HVDLIRSFKVQPSVVIVDMPHMVARHGNIRHP----LQCFDG-DMICGVCGPTPEDTIWDG-----TGGLMCVWCPHSVCYGFHNIPKGEGRNDVFSAIVTRWEKAPKRIIYDFACALGPYCMTREP----LQGWEN-DMACKRCGPSPETVIWDG-----TGGIMVAWCTHSICYGFHCIAESEGRDDVFSALVTRWPIAPKRVVYDFACALGPYCMLREP----LQQFNN-DMCCPRCGPLPQETIWDG-----TGGIMCAWCTHSICYGFHCIPNGEGRNDVFSAMITRWPKAPKVVVYDFACALGPYCLTCEP----LQSLDS-KMQCPTCGPCPKVVIADG-----TGGILILXCSHSICLGFHSIPVAEGRNDVFSAIYTHFPQAPDIIIYDFACQLAQYCLVREA----VQEMRD-DMVCPDCGDAPENLIWDG-----TGGIMCAWCTHSICYGFHCIPTGEGRNDVFSAMVTRWPIAPRRVIYDFACALEPYCMTREP----LIGWGT-VMTCPECGPNPEVVIMDG-----TGGIAGIWCPHGICLAYQVMPTSEGRDDFFSLLKCFFPVPPKVIVYDFACSLATYCMLRDP----RLDRFH-TFKCPICDWRPRVLICDA-----TGGIFTVFCEHGVCYAFFVLPRAEGRNEMYSWMVSFLPRAPEVVVYDFACSLHEYCLNRAP----HLDLPY-KFTCPVCGTSPRRLICDG-----TGGVFTLFCEHRICYAFFILPKAEGRNEVYSWMVSYLEKAPDLVVYDFACNLHEYCMNRAP----LTDHPY-GFFSFIDGFFPLLSSFDG-----EGGYRPD----MFMLRAMFMLRAESPTD-LAVLLLHHVHMPILVITDIMCRVGPIVHDRCQ----LTVRDYNDMICGICGVAPKVEMAQR-----TGGKIYKVCPHQVVCGSKYLVRGESARD-HVDLLASSRHWPPVYVVDMATPVALCADLCYP----- III 149-650aa IV 92-733aa FIG. 4. Identification of conserved regions in divergent Zisupton-related proteins. The four regions of higher conservation (I to IV) and the SWIM domain are shown. Amino acid size variations in nonconserved regions are indicated (size might be affected by models used to predict gene structure from genomic DNA). Identical residues are white in black boxes, similar residues in black on gray boxes. Accession numbers: Ciona intestinalis EAAA01002232, Amphioxus Branchiostoma floridae (Florida lancelet) XP_002610779, Saccoglossus kowalevskii ACQM01108926, Laccaria bicolor ABFE01001864, Coprinopsis cinerea AACS02000001, Serpula lacrymans AEQC01001111, Postia placenta ABWF01004332, Moniliophthora perniciosa ABRE01017974, Cryptococcus neoformans AAEY01000005, Ectocarpus siliculosus CABU01013191, Chlamydomonas reinhardtii ABCN01003575, Volvox carteri ACJH01007750, and Hmgxb3 mouse EDL09783. Zisupton sequences are reminiscent of the structures found in some transposons from the Mutator superfamily (Marquez and Pritham 2010 and references therein). Hence, a distant relationship between Mutator and Zisupton superfamilies appears to be possible. The expression pattern of Zisupton is conserved in the majority of Xiphophorus species tested, with a strong expression in the testis. In addition, initial unpublished data obtained for Zisupton in zebrafish and medaka also point to a preferential expression in gonads, and the acorn worm database sequence was obtained from testis. This is of course not unexpected for a TE, which must be active in the germ line to ensure transmission of new insertions to the next generation. Indeed, preferential activity in male or female germ line has been reported for many elements (Weiner et al. 1986; Jurka et al. 2004). In contrast, TE expression is often downregulated in somatic tissues, probably to restrain damage caused by transposition. The expression of Zisupton in testis is extremely high, especially in X. maculatus. This might be linked to the presence of one copy on the Y chromosome, where sequences preferentially expressed in testis are frequently located (Skaletsky et al. 2003). Depending on alternative splicing, the Zisupton protein can contain (in testis) or not (in all tissues tested) the Ulp1 SUMO protease and the F-box domains. Alternative splicing of transposon mRNA generating different 640 transcripts and eliminating premature stop codons in a tissue-specific manner has already been described for other TEs (Rio 1990; Fujino et al. 2009). In the genus Xiphophorus, Zisupton is extremely conserved between species having diverged 3–20 Ma (Hrbek et al. 2007). In addition, all transposon insertions tested so far were species specific and even population specific within a same species. Taken together, these observations indicate that Zisupton has recently been active in Xiphophorus and has possibly been subject to horizontal transfer. Horizontal transfer has been reported for many elements, especially DNA transposons. This mode of transmission is considered as a way to escape vertical extinction in their original host lineage and to infect nonrelated organisms (Schaack et al. 2010). However, much more sequence information must be obtained for other cellular genes from different Xiphophorus species to assess if interspecific sequence divergence for Zisupton is significantly lower than the divergence between their host genomes. Within vertebrates, Zisupton elements have been widely detected in fish. In contrast, they could not be identified through in silico analysis in any tetrapod lineage. Zisupton-like sequences have also been found in Ciona, amphioxus, and acorn worm. Hence, Zisupton might have been present in ancestral chordates and has been possibly lost during early evolution of the tetrapod lineage. This MBE Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208 I II III IV C2H2 SWIM SAP Ulp1 protease C2H2 SWIM SAP Ulp1 protease SWIM SAP Ulp1 protease C2H2 SWIM SAP ? C2H2 C2H2 SWIM SAP Tetraodon Takifugu C2H2 C2H2 SWIM Amphioxus C2H2 C2H2 SWIM C2H2 SWIM C2H2 SWIM Platyfish C2H2 Zebrafish C2H2 Medaka C2H2 Stickleback C2H2 Takifugu Ciona repetitive motif C2H2 C19 protease SAP Fungi SWIM SAP Chlamydomonas Volvox SWIM SAP OTU domain Ectocarpus HMGXB3 HMGB FIG. 5. Schematic representation of conserved domains in Zisupton-related proteins. Predicted functional motifs within proteins are shown, as well as the four conserved regions, which are represented by vertical gray bars (I to IV, fig. 4). observation confirms that fish genomes contain many more families of TEs than tetrapod genomes and that the evolution of the lineage leading to mammals has undergone a drastic reduction in TE diversity (Volff et al. 2003; Fischer et al. 2005; Böhne et al. 2008). In fish, Zisupton seems to have experienced differential success in different lineages. This element is present with several highly conserved and probably intact copies in Xiphophorus genomes and is represented by 20–30 copies in medaka and zebrafish. In contrast, Zisupton is rare in stickleback and has apparently lost its ability to transpose in puffer fishes. The unique sequence of Tetraodon and one sequence of Fugu are located at the same position in syntenic regions of their respective genomes. These sequences are much shorter than other fish Zisuptons and do not have any TIRs and TSDs. Such a conservation between a transposition- deficient TE in species having diverged approximately 18–30 Ma (Van de Peer 2004) would be quite unusual for simply dead elements. Rather, we suggest that they correspond to bona fide cellular genes derived from a Zisupton transposon. In vertebrates and many other lineages, numerous host genes with important cellular functions have been derived from TEs. This phenomenon is called molecular domestication (Volff 2006 and references therein; Sinzelle et al. 2009). Accordingly, Ka/Ks analysis indicated an evolution under purifying selection, suggesting that puffer fish Zisupton-like orthologous sequences do not correspond to pseudogenes. Interestingly, Zisupton shows phylogenetic relationship with bona fide cellular genes from the vertebrate HMGXB3 family. One unique orthologous HMGXB3 gene is present within syntenic genomic regions in all major vertebrate 641 MBE Böhne et al. · doi:10.1093/molbev/msr208 Ectocarpus Hmgxb3-Tetraodon Hmgxb3-Fugu Hmgxb3-Medaka Hmgxb3-Zebrafish Hmgxb3_Stickleback Ciona2 Ciona1 Ciona3 Hmgxb3_Chicken Hmgxb3_Xenopus Hmgxb3_Mouse Hmgxb3_Human Ciona4 Stickleback Amphioxus 90/83 100/99 56/- Saccoglossus 70/73 40/90 Medaka2 Medaka1 79/73 Fugu Platyfish Zebrafish3 Zebrafish4 Zebrafish2 Zebrafish5 Zebrafish6 Zebrafish1 Tetraodon-dom. Fugu-dom. 100/100 77/56 Cryptococcus 77/99 70/- Postia Serpula2 80/Serpula1 Chlamydomonas3 Serpula4 Chlamydomonas4 Serpula3 Chlamydomonas1 Lacaria2 Moniliophthora2 Chlamydomonas2 Volvox Lacaria1 Moniliophthora1 Lacaria3 Coprinopsis4 Coprinopsis1 Coprinopsis3 Coprinopsis2 FIG. 6. Molecular phylogeny of Zisupton-related proteins. The tree was constructed using maximum likelihood analysis with 100 bootstrap replicates (first values) on concatenated conserved regions shown in figure 4. Bootstrap values in italics (second values) have been obtained using neighbor joining reconstruction (1,000 bootstrap replicates). lineages including fish, amphibians, birds, and mammals. The evolutionary relationship between HMGXB3 and Zisupton remains to be elucidated. Because of the presence of Zisupton sequences in nonvertebrate chordates, hemichordates, fungi, and algae, we propose that HMGXB3 has been derived from a Zisupton TE via an event of molecular domestication in a common ancestor of fish and tetrapods at least 450 Ma. Alternatively, the Zisupton transposon might have been formed from an HMGXB3-like gene and transmitted to more divergent organisms through horizontal transfer. Consistent with a fish-specific acquisition of the Cterminal region, the Ulp1 SUMO protease/F-box region was detected only in fish Zisupton sequences. The amphioxus sequence contains a C19 protease, also known as ubiquitin carboxyl-terminal hydrolase. This protease is involved in the processing of poly-ubiquitin precursors and ubiquitinated proteins, a function similar to that of Ulp1 in the SUMO pathway (Jentsch et al. 1991; Barrett and Rawlings 2001). In the acorn worm, the protease domain is an OTU protease (cysteine protease) (Makarova et al. 2000). The evolution of TEs is often modular; different regions in one element can have different evolutionary origins (Lerat et al. 1999; Capy and Maisonhaute 2002). Accordingly, Zisupton might have captured an Ulp1 SUMO protease gene during evolution, possibly early in the fish lineage. The closest cellular proteases 642 related to Zisupton are SENP proteins, sentrin-specific proteases (sentrin is another name for SUMO). Sentrin proteases catalyze the removal of SUMO from its substrates as well as its activating cleavage from a precursor form (Drag and Salvesen 2008; Yeh 2009). Hence, Zisupton transposons might have recruited a sentrin-specific protease, together with a protein–protein interaction domain called F-box, which is present in the C-terminal region too. F-box proteins are implicated in the ubiquitin pathway in the ligation step of ubiquitin (Skowyra et al. 1997). The C-terminal part of the Zisupton protein might interfere with ubiquitin/SUMO posttranslational protein modification and enhance the transposition efficiency of the element, for example, through the inhibition of protein degradation in the proteasome. Accordingly, ubiquitin/SUMO pathways have been shown to modulate the activity of several TEs (Kang et al. 1992; Menees and Sandmeyer 1996). To conclude, Zisupton represents a new type of DNA transposon active in fish and detected in various divergent organisms. This element does not share any obvious similarity with already described TEs. Enzymes catalyzing its transposition must be now characterized at the functional level. Zisupton distribution, evolutionary dynamics, and effects on genomes outside of poeciliid fish must be further analyzed. Of particular interest are its potential ability to be transferred horizontally between species and to serve as a source for the Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208 formation of novel genes, the function of which is still to be elucidated. From all these features, Zisupton has the potential of a powerful agent of genomic diversity and evolutionary novelties in fish and other organisms. Acknowledgments This work was supported by a European Mobility PhD fellowship from the French Ministry of National Education, Research and Technology to A.B. and by grants from the BioFuture program of the Bundesministerium für Bildung und Forschung (BMBF), the Agence Nationale de la Recherche (ANR), the Institut National de la Recherche Agronomique (INRA, département PHASE), the Centre National de la Recherche Scientifique (CNRS), and the Fondation pour la Recherche Médicale (FRM) to J.-N.V. References Aravind L, Koonin EV. 2000. SAP—a putative DNA-binding motif involved in chromosomal organization. Trends Biochem Sci. 25:112–114. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. 2010. Current protocols in molecular biology. Hoboken (NJ): Wiley. Banerjee R, Dubois DY, Gauthier J, Lin SX, Roy S, Lapointe J. 2004. The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm. Eur J Biochem. 271:724–733. Bao W, Kapitonov VV, Jurka J. 2010. Ginger DNA transposons in eukaryotes and their evolutionary relationships with long terminal repeat retrotransposons. Mob DNA. 1:3. Barrett AJ, Rawlings ND. 2001. Evolutionary lines of cysteine peptidases. Biol Chem. 382:727–733. Bennetzen JL. 2000. Transposable element contributions to plant gene and genome evolution. Plant Mol Biol. 42:251–269. Böhne A, Brunet F, Galiana-Arnoux D, Schultheis C, Volff JN. 2008. Transposable elements as drivers of genomic and biological diversity in vertebrates. Chromosome Res. 16:203–215. Böhne A, Schultheis C, Galiana-Arnoux D, et al. (20 co-authors). 2009. Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: towards the identification of a new type of master sexual regulator in vertebrates. Integr Zool. 4:277–284. Bourque G. 2009. Transposable elements in gene regulation and in the evolution of vertebrate genomes. Curr Opin Genet Dev. 19:607–612. Brayer KJ, Segal DJ. 2008. Keep your fingers off my DNA: proteinprotein interactions mediated by C2H2 zinc finger domains. Cell Biochem Biophys. 50:111–131. Brown RS. 2005. Zinc finger proteins: getting a grip on RNA. Curr Opin Struct Biol. 15:94–98. Cai M, Qiu D, Yuan T, Ding X, Li H, Duan L, Xu C, Li X, Wang S. 2008. Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance. Plant Cell Environ. 31:86–96. Capy P, Maisonhaute C. 2002. Acquisition/loss of modules: the construction set of transposable elements. Genetika. 38:594–601. Craig KL, Tyers M. 1999. The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction. Prog Biophys Mol Biol. 72:299–328. Cummings ME, Gelineau-Kattner R. 2009. The energetic costs of alternative male reproductive strategies in Xiphophorus nigrensis. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 195:935–946. Drag M, Salvesen GS. 2008. DeSUMOylating enzymes—SENPs. IUBMB Life. 60:734–742. MBE Edgar RC. 2004a. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 5:113. Edgar RC. 2004b. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792–1797. Ezaz T, Stiglec R, Veyrunes F, Marshall Graves JA. 2006. Relationships between vertebrate ZW and XY sex chromosome systems. Curr Biol. 16:736–743. Fernandez AA, Fernandez LR, Toth L. 2008. Head over heels: an examination of a possible mating signal in female swordtails, Xiphophorus cortezi. Anim Behav. 76:1073–1081. Feschotte C, Pritham EJ. 2005. Non-mammalian c-integrases are encoded by giant transposable elements. Trends Genet. 21:551–552. Fischer C, Bouneau L, Coutanceau JP, Weissenbach J, OzoufCostaz C, Volff JN. 2005. Diversity and clustered distribution of retrotransposable elements in the compact genome of the pufferfish Tetraodon nigroviridis. Cytogenet Genome Res. 110:522–536. Froschauer A, Körting C, Katagiri T, Aoki T, Asakawa S, Shimizu N, Schartl M, Volff JN. 2002. Construction and initial analysis of bacterial artificial chromosome (BAC) contigs from the sexdetermining region of the platyfish Xiphophorus maculatus. Gene 295:247–254. Fujino K, Matsuda Y, Sekiguchi J. 2009. Transcriptional activity of rice autonomous transposable element Dart. J Plant Physiol. 166:1537–1543. Gordon M. 1927. The genetics of a viviparous top-minnow platypoecilus; the inheritance of two kinds of melanophores. Genetics 12:253–283. Gray YH. 2000. It takes two transposons to tango: transposableelement-mediated chromosomal rearrangements. Trends Genet. 16:461–468. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 59:307–321. Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 52:696–704. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 41:95–98. Haun WJ, Danilevskaya ON, Meeley RB, Springer NM. 2009. Disruption of imprinting by mutator transposon insertions in the 5’ proximal regions of the Zea mays Mez1 locus. Genetics 181:1229–1237. Hoen DR, Park KC, Elrouby N, Yu Z, Mohabir N, Cowan RK, Bureau TE. 2006. Transposon-mediated expansion and diversification of a family of ULP-like genes. Mol Biol Evol. 23:1254–1268. Hrbek T, Seckinger J, Meyer A. 2007. A phylogenetic and biogeographic perspective on the evolution of poeciliid fishes. Mol Phylogenet Evol. 43:986–998. Iuchi S. 2001. Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci. 58:625–635. Jackson PK, Eldridge AG. 2002. The SCF ubiquitin ligase: an extended look. Mol Cell. 9:923–925. Jentsch S, Seufert W, Hauser HP. 1991. Genetic analysis of the ubiquitin system. Biochim Biophys Acta. 1089:127–139. Jurka J, Kapitonov VV, Kohany O, Jurka MV. 2007. Repetitive sequences in complex genomes: structure and evolution. Annu Rev Genomics Hum Genet. 8:241–259. Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J. 2005. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet Genome Res. 110:462–467. 643 Böhne et al. · doi:10.1093/molbev/msr208 Jurka J, Kohany O, Pavlicek A, Kapitonov VV, Jurka MV. 2004. Duplication, coclustering, and selection of human Alu retrotransposons. Proc Natl Acad Sci U S A. 101:1268–1272. Kallman KD, Kazianis S. 2006. The genus Xiphophorus in Mexico and central America. Zebrafish 3:271–285. Kang XL, Yadao F, Gietz RD, Kunz BA. 1992. Elimination of the yeast RAD6 ubiquitin conjugase enhances base-pair transitions and G.C––T.A transversions as well as transposition of the Ty element: implications for the control of spontaneous mutation. Genetics 130:285–294. Kapitonov VV, Jurka J. 2001. Rolling-circle transposons in eukaryotes. Proc Natl Acad Sci U S A. 98:8714–8719. Kapitonov VV, Jurka J. 2006. Self-synthesizing DNA transposons in eukaryotes. Proc Natl Acad Sci U S A. 103:4540–4545. Kapitonov VV, Jurka J. 2007. Helitrons on a roll: eukaryotic rollingcircle transposons. Trends Genet. 23:521–529. Kapitonov VV, Jurka J. 2008. Universal classification of eukaryotic transposable elements implemented in Repbase. Nat Rev Genet. 9:411–412. Kohany O, Gentles AJ, Hankus L, Jurka J. 2006. Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor. BMC Bioinformatics. 7:474. Kosswig C. 1928. Über Kreuzungen zwischen Teleostiern Xiphophorus hellerii und Platypoecilius maculatus. Z Indukt Abstamm Vererbungsl. 47:150–158. Lampert KP, Schmidt C, Fischer P, Volff JN, Hoffmann C, Muck J, Lohse MJ, Ryan MJ, Schartl M. 2010. Determination of onset of sexual maturation and mating behavior by melanocortin receptor 4 polymorphisms. Curr Biol. 20:1729–1734. Lerat E, Brunet F, Bazin C, Capy P. 1999. Is the evolution of transposable elements modular? Genetica 107:15–25. Lin R, Teng Y, Park HJ, Ding L, Black C, Fang P, Wang H. 2008. Discrete and essential roles of the multiple domains of Arabidopsis FHY3 in mediating phytochrome A signal transduction. Plant Physiol. 148:981–992. Lippman Z, Gendrel AV, Black M, et al. (14 co-authors). 2004. Role of transposable elements in heterochromatin and epigenetic control. Nature 430:471–476. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. Makarova KS, Aravind L, Koonin EV. 2000. A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae. Trends Biochem Sci. 25:50–52. Makarova KS, Aravind L, Koonin EV. 2002. SWIM, a novel Znchelating domain present in bacteria, archaea and eukaryotes. Trends Biochem Sci. 27:384–386. Marı́n I. 2010. GIN transposons: genetic elements linking retrotransposons and genes. Mol Biol Evol. 27:1903–1911. Marquez CP, Pritham EJ. 2010. Phantom, a new subclass of Mutator DNA transposons found in insect viruses and widely distributed in animals. Genetics 185:1507–1517. Meierjohann S, Schartl M. 2006. From Mendelian to molecular genetics: the Xiphophorus melanoma model. Trends Genet. 22:654–661. Menees TM, Sandmeyer SB. 1996. Cellular stress inhibits transposition of the yeast retrovirus-like element Ty3 by a ubiquitindependent block of virus-like particle formation. Proc Natl Acad Sci U S A. 93:5629–5634. Meyer A, Salzburger W, Schartl M. 2006. Hybrid origin of a swordtail species (Teleostei: Xiphophorus clemenciae) driven by sexual selection. Mol Ecol. 15:721–730. Muffato M, Louis A, Poisnel CE, Roest Crollius H. 2010. Genomicus: a database and a browser to study gene synteny in modern and ancestral genomes. Bioinformatics 26:1119–1121. Nelson JS. 2006. Fishes of the world. 4th ed. Hoboken (NJ): John Wiley & Sons, Inc. 644 MBE Nishito Y, Hasegawa M, Inohara N, Núñez G. 2006. MEX is a testisspecific E3 ubiquitin ligase that promotes death receptorinduced apoptosis. Biochem J. 396:411–417. Notredame C, Higgins D, Heringa J. 2000. T-Coffee: a novel method for multiple sequence alignments. J Mol Biol. 302:205–217. Offen N, Meyer A, Begemann G. 2009. Identification of novel genes involved in the development of the sword and gonopodium in swordtail fish. Dev Dyn. 238:1674–1687. Papadopoulos JS, Agarwala R. 2007. COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics 23:1073–1079. Parks CL, Chang LS, Shenk T. 1991. A polymerase chain reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region. Nucleic Acids Res. 19:7155–7160. Pritham EJ, Putliwala T, Feschotte C. 2007. Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses. Gene 390:3–17. Rio DC. 1990. Molecular mechanisms regulating Drosophila P element transposition. Annu Rev Genet. 24:543–578. Rosenfeld R, Margalit H. 1993. Zinc fingers: conserved properties that can distinguish between spurious and actual DNA-binding motifs. J Biomol Struct Dyn. 11:557–570. Schaack S, Gilbert C, Feschotte C. 2010. Promiscuous DNA: horizontal transfer of transposable elements and why it matters for eukaryotic evolution. Trends Ecol Evol. 25:537–546. Schultheis C, Zhou Q, Froschauer A, et al. (22 co-authors). 2006. Molecular analysis of the sex-determining region of the platyfish Xiphophorus maculatus. Zebrafish 3:299–309. Sinzelle L, Izsvak Z, Ivics Z. 2009. Molecular domestication of transposable elements: from detrimental parasites to useful host genes. Cell Mol Life Sci. 66:1073–1093. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et al. (40 co-authors). 2003. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423:825–837. Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW. 1997. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91:209–219. Smit AF. 2002. Initial survey of interspersed repeats in Takifugu rubripes. Repbase Rep 2:74. Suzuki R, Shindo H, Tase A, Kikuchi Y, Shimizu M, Yamazaki T. 2009. Solution structures and DNA binding properties of the Nterminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa. Proteins 75:336–347. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 24:1596–1599. Thomas JO. 2001. HMG1 and 2: architectural DNA-binding proteins. Biochem Soc Trans. 29:395–401. Van de Peer Y. 2004. Tetraodon genome confirms Takifugu findings: most fish are ancient polyploids. Genome Biol. 5:250. van Leeuwen H, Monfort A, Puigdomenech P. 2007. Mutator-like elements identified in melon, Arabidopsis and rice contain ULP1 protease domains. Mol Genet Genomics. 277:357–364. Volff JN. 2005. Genome evolution and biodiversity in teleost fish. Heredity 94:280–294. Volff JN. 2006. Turning junk into gold: domestication of transposable elements and the creation of new genes in eukaryotes. Bioessays 28:913–922. Volff JN, Bouneau L, Ouzouf-Costaz C, Fischer C. 2003. Diversity of retrotransposable elements in compact pufferfish genomes. Trends Genet. 19:674–678. Volff JN, Körting C, Altschmied J, Duschl J, Sweeney K, Wichert K, Froschauer A, Schartl M. 2001. Jule from the fish Xiphophorus is the first complete vertebrate Ty3/Gypsy retrotransposon from the Mag family. Mol Biol Evol. 18:101–111. Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208 Volff JN, Körting C, Froschauer A, Sweeney K, Schartl M. 2001. NonLTR retrotransposons encoding a restriction enzyme-like endonuclease in vertebrates. J Mol Evol. 52:351–360. Volff JN, Körting C, Sweeney K, Schartl M. 1999. The non-LTR retrotransposon Rex3 from the fish Xiphophorus is widespread among teleosts. Mol Biol Evol. 16:1427–1438. Weiner AM, Deininger PL, Efstratiadis A. 1986. Nonviral retroposons: genes, pseudogenes, and transposable elements generated by the reverse flow of genetic information. Annu Rev Biochem. 55:631–661. Wicker T, Sabot F, Hua-Van A, et al. (13 co-authors). 2007. A unified classification system for eukaryotic transposable elements. Nat Rev Genet. 8:973–982. MBE Yeh ET. 2009. SUMOylation and De-SUMOylation: wrestling with life’s processes. J Biol Chem. 284:8223–8227. Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, Yu J. 2006. KaKs Calculator: calculating Ka and Ks through model selection and model averaging. Genomics Proteomics Bioinformatics 4:259–263. Zhou Q, Braasch I, Froschauer A, Böhne A, Schultheis C, Schartl M, Volff JN. 2010. A novel marker for the platyfish (Xiphophorus maculatus) W chromosome is derived from a Polinton transposon. J Genet Genomics. 37:181–188. Zhou Q, Froschauer A, Schultheis C, Schmidt C, Bienert GP, Wenning M, Dettai A, Volff JN. 2006. Helitron transposons on the sex chromosomes of the platyfish Xiphophorus maculatus and their evolution in animal genomes. Zebrafish 3:39–52. 645