Multiple Lineages of the Non-LTR Retrotransposon Rex1 with Varying Success in Invading Fish Genomes

Jean-Nicolas Volff, Cornelia Körting and Manfred Schartl

Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg, Germany


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Rex1, together with the related Babar elements, represents a new family of non-long-terminal-repeat (non-LTR) retrotransposons from fish, which might be related to the CR1 clade of LINE elements. Rex1/Babar retrotransposons encode a reverse transcriptase and an apurinic/apyrimidinic endonuclease, which is very frequently removed by incomplete reverse transcription. Different Rex1 elements show a conserved terminal 3' untranslated region followed by oligonucleotide tandem repeats of variable size and sequence. Phylogenetic analysis revealed that Rex1 retrotransposons were frequently active during fish evolution. They formed multiple ancient lineages, which underwent several independent and recent bursts of retrotransposition and invaded fish genomes with varying success (from <5 to 500 copies per haploid genome). At least three of these ancient Rex1 lineages were detected within the genome of poeciliids. One lineage is absent from some poeciliids but underwent successive rounds of retrotransposition in others, thereby increasing its copy number from <10 to about 200. At least three ancient Rex1 lineages were also detected in the genome project fish Fugu rubripes. Rex1 distribution within one of its major lineages is discontinuous: Rex1 was found in all Acanthopterygii (common ancestor in the main teleost lineage approximately 90 MYA) and in both European and Japanese eels (divergence from the main teleost lineage about 180 MYA) but not in trout, pike, carp, and zebrafish (divergence 100–120 MYA). This might either result from frequent loss or rapid divergence of Rex1 elements specifically in some fish lineages or represent one of the very rare examples of horizontal transfer of non-LTR retrotransposons. This analysis highlights the dynamics and complexity of retrotransposon evolution and the variability of the impact of retrotransposons on vertebrate genomes.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Fishes, the most diverse group of living vertebrates, should possess efficient genomic tools for the generation of genetic variability. Due to their mobility and their repetitive nature, transposable elements are a source of variation in animals and other organisms (Kidwell and Lisch 1997Citation ; Kazazian and Moran 1998Citation ). Fish genomes contain all types of known transposable elements: classical DNA transposons (Izsvák, Ivics, and Hackett 1995Citation ; Koga et al. 1996Citation ), miniature inverted-repeat transposable elements (Izsvák, Ivics, and Hackett 1997Citation ) and retroelements including long terminal repeat (LTR) retrotransposons (Flavell and Smith 1992Citation ; Britten et al. 1995Citation ; Tristem et al. 1995Citation ; Poulter and Butler 1998Citation ; Miller et al. 1999Citation ), non-LTR retrotransposons (also called long interspersed elements [LINEs]) (Winkfein et al. 1988Citation ; Ohshima et al. 1996Citation ; Okada et al. 1997Citation ; Duvernell and Turner 1998Citation ; Poulter, Butler, and Ormandy 1999Citation ; Volff et al. 1999Citation ), and short interspersed elements (SINEs; Izsvák et al. 1996Citation ). While DNA transposons move directly as DNA molecules from one genomic site into another, retroelements transpose via an RNA intermediate. Unlike SINE elements, complete versions of LTR and non-LTR retrotransposons encode their own enzymatic machinery for transposition, including a reverse transcriptase (RT). Non-LTR retrotransposons do not possess flanking long duplicated sequences like those found in LTR retrotransposons and are frequently truncated at their 5' end due to incomplete reverse transcription of their RNA.

Almost nothing is known about the evolution and the genomic impact of fish retroelements. Only a small number of mutant phenotypes due to retroelement-mediated gene disruption have been reported to date in fishes (Izsvák et al. 1996Citation ; Schartl et al. 1999Citation ). Several evolutionary studies have already been performed on fish retroelements (e.g., Hamada et al. 1998Citation ; Duvernell and Turner 1999Citation ), but, to our knowledge, no extensive survey including representative species of the bony fish lineage has been conducted to understand retrotransposon evolution on a larger evolutionary scale. The teleost Xiphophorus (poeciliid) is an established model for melanoma research, analysis of sex determination, and many questions of evolutionary biology (Kallman 1984Citation ; Ryan and Wagner 1987Citation ; Meyer and Lydeard 1993Citation ; Schartl 1995Citation ). We describe here the non-LTR retrotransposon Rex1 from the platyfish Xiphophorus maculatus and its complex evolutionary dynamics in teleost genomes.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Fishes
The following fishes were from stocks maintained at the University of Würzburg: Xiphophorus maculatus (Rio Jamapa, Rio Usumacinta), Xiphophorus helleri (Rio Lantecilla), Xiphophorus nezahualcoyotl (Rio El Salto), Xiphophorus montezumae (Cascadas de Tamasopo), Gambusia affinis (Pena Blanca), Poeciliopsis gracilis (Rio Jamapa), Heterandria bimaculata (Tierra Blanca) and Heterandria formosa (Fort Lauderdale), Phallichthys amates (aquarium stock), Poecilia mexicana (Media Luna), Poecilia latipinna (Key Largo) and Poecilia formosa (Tampico), Girardinus metallicus (aquarium stock) and Girardinus falcatus (aquarium stock), Fundulus sp. (Laguna de Labradores), Oryzias latipes (medakafish strain HB32c), Danio rerio (zebrafish strain m14), and Cichlasoma labridens (Cascadas de Tamasopo). Rainbow trout (Oncorhynchus mykiss), pike (Esox lucius), common carp (Cyprinus carpio), European eel (Anguilla anguilla), and sturgeon (Acipenser sturio) were obtained from a local fish farm near Würzburg. Hemichromis bimaculatus (Comoe National Park, Ivory Coast) was a gift from K. Mody (University of Würzburg). Genomic DNAs from Nile tilapia (Oreochromis niloticus, Göttingen, Germany) and Japanese eel (Anguilla japonica) were obtained from S. Chen (University of Würzburg, Germany, and Huanghai Fisheries Research Institute, Qingdao, China) and from Y. Nakatsuru and T. Ishikawa (University of Tokyo, Japan), respectively. Genomic DNA and organs from Battrachocottus baikalensis were a gift from S. Kirilchik and M. Grachev (Institute of Limnology, Irkutsk, Russia). The phylogeny of these fishes (according to http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html) is given in figure 1 and is supported by the analysis of the nucleotide sequence of the growth hormone gene (Venkatesh and Brenner 1997Citation ).



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Fig. 1.—Phylogenetic relationship of fish species included in this study

 
DNA Manipulations
Genomic DNA was isolated as described by Schartl et al. (1996)Citation . The genomic library of X. maculatus consisted of 35–45-kb inserts cloned into cosmid Lawrist7 (Burgtorf et al. 1998Citation ). Sequence encoding RT domains 3–7 (526 nt in length in Rex1-XimJ) was amplified by PCR as described (Volff et al. 1999Citation ) using primer RTX1-F1 (5'-ttctccagtgccttcaacacc-3') with either RTX1-R1 (5'-ttccttaaaaaatagagtctgctc-3', from Rex1-XimJ) or RTX1-R3 (5'-tccctcagcagaaagagtctgctc-3', from Rex1-Bab). PCR products were cloned and sequenced on both strands. When identical sequences were isolated from a fish, only one was retained for further analysis. Cosmid sequence was obtained by transposon mutagenesis (Fischer et al. 1996Citation ). Sequencing, Southern blot analysis, and copy number estimation by quantitative slot-blotting were done as described (Volff et al. 1999Citation ). Filters were washed with 2 x SSC, 1% SDS at 50°C.

DNA Sequence Analysis
Nucleotide sequences were analyzed using the GCG Wisconsin package (version 10.0, Genetics Computer Group, Madison, Wis.). Multiple sequence alignments used for phylogenetic analysis were generated using PileUp. Pairwise evolutionary distances between aligned sequences were determined with the Distances program of GCG using the Kimura (1980)Citation two-parameter distance method. The pairwise numbers of synonymous (Ks) and non-synonymous substitutions (Ka) per site between two aligned sequences was estimated with the Diverge program of GCG. This program is based on the method described by Li, Wu, and Luo (1985), as modified by Li (1993)Citation and Pamilo and Bianchi (1993)Citation , and applies Kimura's (1980)Citation two-parameter method. Phylogenetic analyses were done with PAUP* (Swofford 1989Citation ) as part of the GCG package. Trees were generated through a heuristic search using maximum parsimony and distance (minimum evolution) as optimality criteria (bootstrap analysis, 100 replicates). Neighbor-joining bootstrap analysis (1,000 replicates) was also performed.

Data Deposition
Partial Rex1 sequences reported in this paper can be found in the EMBL database under accession numbers AJ288431–AJ288486. Rex1-XimJ sequence from the genomic cosmid bank has been deposited in GenBank under accession number AF155728. The sequence alignment used to generate Rex1 phylogeny can be found in the EMBL nucleotide sequence database under accession number DS42497. Fugu and Tetraodon consensus sequences are available on request.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Rex1 and Babar Define a New Family of Fish Non-LTR Retrotransposons
Analysis of cosmid D18 097, which contains an insert from the Y sex chromosome of X. maculatus, revealed a sequence encoding a putative product with similarities to RTs of non-LTR retrotransposons (figs. 2 and 3 ). Additional endonuclease domain, flanking open reading frame, and LTR sequences could not be found. Hence, this sequence is a truncated copy of a non-LTR retrotransposon that we called Rex1-XimJ (Retroelement Xiphophorus 1–Xiphophorus maculatus Rio Jamapa). Using Rex1-XimJ as query, we identified other Rex1 elements in public sequence databases (table 1 and figs. 2 and 3 ). Rex1-Bab was detected in a genomic sequence from the fish B. baikalensis. The Rex1-Bab RT-encoding region is disrupted in domain 8 by a 470-nt sequence, which seems to be neither a tRNA-derived short interspersed element nor a miniature inverted-repeat transposable element. This sequence is flanked by a tetranucleotide duplication, which may correspond to the duplication of the target site sequence, and shows at one end four tandem repeats of the hexanucleotide GATTCT (in one case, A is replaced by G). Tandem oligonucleotide repeats are found at the 3' end of some non-LTR retrotransposons. Hence, the insertion disrupting Rex1-Bab may correspond to an extremely truncated non-LTR retrotransposon. Using sequences from the genome project of the Japanese pufferfish Fugu rubripes (Elgar et al. 1996Citation ; http://fugu.hgmp.mrc.ac.uk), at least three distinct phylogenetic groups of sequences were identified, leading to the reconstruction of three partial Rex1 variants called Rex1-FurA, Rex1-FurB, and Rex1-FurC. Analysis of an additional unique sequence, which was too short to be included in this study, even suggested the presence of a fourth variant in the genome of F. rubripes (data not shown). One extremely truncated copy of Rex1-FurA is present in one intron of the F. rubripes gene for the D3-like dopamine receptor (X80176). A truncated FurC element is located between two V (variable) segments of the F. rubripes T-cell receptor alpha-chain gene (AF110525). Inspection of sequences from the genome project of the related freshwater pufferfish Tetraodon nigroviridis (http://www.genoscope.cns.fr) revealed only one Rex1 variant, which was called Rex1-Ten. One strongly truncated copy of Rex1-Ten is present in the intergenic spacer of a 28S rRNA gene of T. nigroviridis (AJ270047).



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Fig. 2.—Comparison of Rex1 and Babar reverse transcriptase (RT) sequences with those of other non-LTR retrotransposons (upper panel), and comparison of the C-terminal domains of Rex1 and Babar elements (lower panel). RT conserved domains are given according to Malik, Burke, and Eickbush (1999)Citation and Volff et al. (1999)Citation . Identical residues are in black, and conservative substitutions are in gray. Rex1 and Babar RT sequences are conceptual translation products of sequences described in table 1 . Other RT sequences are CR1 from Gallus gallus (U88211), Maui from Fugu rubripes (AF086712), Jockey, R1Dm, and I from Drosophila melanogaster (P21328, X51968, and M14954), and Tad1 from Neurospora crassa (L25662)

 

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Table 1 Rex1 and Babar Sequences Used in this Work

 
Another Rex1-related non-LTR retrotransposon was found in the genomic sequence of B. baikalensis (U18939) about 200 nt upstream from Rex1-Bab (table 1 and figs. 2 and 3 ) and was called Babar-Bab (for Batrachocottus baikalensis retrotransposon). Babar retrotransposons were also detected in the insulin-like growth factor I gene of Oncorhynchus keta (Babar-Onk from chum salmon [AF063216]; both the 3' and the 5' ends were missing) and reconstructed from sequences of genome project databases of F. rubripes (Babar-Fur) and T. nigroviridis (Babar-Ten) (table 1 and figs. 2 and 3 ). Preliminary analysis of additional short sequences present in these databases suggested the presence of several Babar variants in F. rubripes and T. nigroviridis (not shown). Partial Babar elements were also identified in database genomic sequences from Gadus morus (Atlantic cod, immunoglobulin light chain gene; AF104899) and from the salmonids Salmo salar (atlantic salmon, immunoglobulin M heavy chain gene; Y12392) and Salvelinus namaycush (lake trout, Tc1-like transposon-containing sequence; AF076967) (not shown). Babar retrotransposons are closely related to but phylogenetically clearly separated from the group of Rex1 elements (fig. 3 ).



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Fig. 3.—Phylogenetic position of Rex1 and Babar reverse transcriptase (RT) sequences in the evolutionary tree of non-LTR retrotransposons. Amino acid similarities with Rex1-XimJ RT are given in %. Expected values obtained by comparing Rex1-XimJ RT amino-acid sequence with the "nr database" (nonredundant GenBank CDS translations + PDB + SwissProt + PIR + PRF) on the ncbi/nlm/nih advanced Blast server (http://www.ncbi.nlm.nih.gov/BLAST) are given in brackets. Analysis was performed used the full length of the RT shown in figure 2 . Minimal and maximal bootstrap values obtained with different types of analysis are given. The 50% majority-rule consensus tree was rooted on the SW1/L1H clade (Malik, Burke, and Eickbush 1999Citation ). The more divergent clades CRE, R2, and R4 are not shown. At least one representative of each of the eight other clades was included: Q from Anopheles gambiae (U03849), Sam6 and Rte1 from Caenorhabditis elegans (Z82275-AF054983), PsCR1 from Platemys spixii (AB005891), SR1 from Schistosoma mansoni (U66331), Juan and Lian from Aedes aegypti (M95171-U87543), CgT1 from Glomerella cingulata (L76169), RT1 from Aedes gambiae (M93690), Rex3 from Xiphophorus maculatus (AF125982), SW1 from Oryzias latipes (AF055640), and Line-1 from humans (L1H, U93574). Other elements are described in the legend of figure 2

 
Phylogenetic analysis of the putative RT amino acid sequences (fig. 3 ) revealed that Rex1 and Babar retrotransposons cannot be assigned to one of the known clades of non-LTR retrotransposons (Malik, Burke, and Eickbush 1999Citation ). Nevertheless, evaluation of percentages of similarity and expected values suggested a slightly closer relationship to known members of the CR1 clade of non-LTR retrotransposons (fig. 3 ).

No endonuclease-encoding domain could be detected upstream (or downstream) of the RT-encoding sequence in Rex1-XimJ or in almost all other related sequences from public databases. This suggested a frequent 5' truncation of Rex1/Babar elements, probably due to incomplete reverse transcription. However, assembling sequences from the T. nigroviridis genome project showed that Rex1-Ten carries an apurinic/apyrimidinic (AP) endonuclease-encoding sequence located upstream of the RT-encoding domain (fig. 4 ). Similar AP endonuclease sequences were identified in F. rubripes cosmids containing nontruncated Rex1-FurA and Rex1-FurB RT sequences (fig. 4 ). As the sequencing of these Fugu cosmids had not been completed, Rex1-FurA and Rex1-FurB endonuclease sequences could not be assembled into a single contig with their respective RT sequences. A corrupted AP endonuclease sequence could be identified upstream from Babar-Bab too (fig. 4 ). Hence, complete versions of Rex1 and Babar retrotransposons encode an AP endonuclease. This confirms the scenario proposed for the evolution of non-LTR retrotransposons (Malik, Burke, and Eickbush 1999Citation ). Comparing Rex1-Ten endonuclease amino acid sequences with the "nr database" (nonredundant GenBank CDS translations + PDB + SwissProt + PIR + PRF) on the ncbi/nlm/nih advanced Blast server (http://www.ncbi.nlm.nih.gov/BLAST) revealed significant similarities only to AP endonucleases encoded by CR1-related retrotransposons, again suggesting a phylogenetic relationship of the Rex1/Babar family with the CR1 clade (not shown). Rex1 and Babar retrotransposons might also encode an additional open reading frame, as observed for most other non-LTR retrotransposons. Such an open reading frame was not detected in this work, probably because of the high frequency of 5' truncation of Rex1/Babar elements, and might be revealed by completion of the Fugu and Tetraodon genome projects.



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Fig. 4.—Comparison of the apurinic-apyrimidinic endonuclease domain of Rex1 and Babar with that of other non-LTR retrotransposons. Identical residues are in black, and conservative substitutions are in gray. Residues generally conserved among non-LTR retrotransposon apurinic-apyrimidinic endonucleases are indicated with asterisks. Accession numbers are given in the legend of figure 2

 
A certain degree of amino acid conservation was detected within the C-terminal domain flanking the RT region of Rex1 and Babar elements, suggesting a functional role of this domain (fig. 2 , lower panel). Nevertheless, neither obvious conservation with the corresponding domain of other retrotransposons nor known protein motifs could be identified in these sequences.

Comparison of Rex1 untranslated regions revealed a segment with a high degree of conservation in the 3' terminal region about 200–300 nt downstream of the RT/C-terminal domain stop codon (fig. 5 ). This observation, which is reminiscent of that reported for HER1/HE1 elements in elasmobranchs (Ogiwara et al. 1999Citation ), suggests that this region may serve as a recognition site for Rex1-encoded reverse transcriptase.



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Fig. 5.—Comparison of the 3' untranslated regions of Rex1 retrotransposons. Sequences start from the stop codon of the Rex1 polyprotein. Nucleotides identical in at least four sequences are shown in black. The 3' ends of Rex1-FurB, as well as those of Babar retrotransposons, could not be determined unambiguously and were therefore not included in this analysis

 
Rex1 retrotransposons end with oligonucleotide tandem repeats of variable size (5–7 nt in length) and variable sequence directly flanking the conserved 3'-terminal region (fig. 5 ). The Rex1-Ten repeat sequence is identical to that of Rex1-XimJ (CTATT) but different from that of the more related (see below) Rex1-FurA and Rex1-Bab elements. It is tempting to speculate that repeat sequence might be able to influence the preference for certain target integration sites. Elements with new terminal repeats might have been selected because they were able to integrate into different sequences and to colonize new genomic niches.

Multiple Rex1 Lineages in Teleosts
PCR amplification of the sequence encoding RT domains 3–7 was performed for various fishes using primers matching either Rex1-XimJ or Rex1-Bab. PCR products were obtained with at least one primer combination from all DNA sources except A. sturio, C. carpio, D. rerio, and O. mykiss (E. lucius has not been tested). Some partial Rex1 sequences were used as probes in Southern blot hybridization to investigate Rex1 distribution (Fig. 6 ) or in quantitative slot-blotting to determine Rex1 copy number (data not shown). Sequence names and origins, as well as numbers of sequenced PCR products, distinct sequences, and distinct sequences with a corrupted partial RT open reading frame, are given in table 1 .



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Fig. 6.—Southern blot analysis of the distribution of Rex1 variants in bony fishes. Probes: A, Rex1-XimJ; B, Rex1-Pog; C, Rex1-Orl2; D, Rex1-Orn; E, Rex1-Ana. Genomic DNA was cut with EcoRI. Signals in Oncorhynchus mykiss (A) probably result from a higher hybridization background due to the larger amount of DNA combined with overexposure of the filter

 
Using Babar nucleotide sequences as an outgroup according to figure 3 , the phylogeny of Rex1 sequences can be explained by the presence of four major ancient lineages (lineages 1–4; fig. 7 ). Lineage 1 is more related to lineage 2, and lineage 3 is more related to lineage 4. Three of these lineages were detected in the Fugu rubripes genome project database (FurA, FurB, and FurC).



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Fig. 7.—Phylogenetic analysis of the partial nucleotide sequence of the reverse transcriptase–encoding region of Rex1 elements. The 50% majority-rule consensus tree was rooted on Babar sequences. Minimal and maximal bootstrap values obtained with different types of analysis are given. 1–4 show the four Rex1 main lineages. Abbreviations for sequences are given in table 1

 
Lineage 1 contains sequences from the poeciliid P. gracilis (Pog) and the F. rubripes variant C (FurC). Using a Pog element as a probe, lineage 2 was found to be present in all poeciliids tested with less than 10 copies per haploid genome (fig. 6B ). With the same probe, no signal could be observed in more divergent fishes.

Lineage 2 contains only sequences isolated from poeciliids, including Xiphophorus (XimJ, XimU, and Xih), G. affinis (Gaa), P. amates (Pha), and P. mexicana (Pom) (fig. 7 ). Using a XimJ sequence as a probe, lineage 2 elements were also detected by Southern blot hybridization in other poeciliids, including H. bimaculata, H. formosa, P. latipinna, and P. formosa (fig. 6A ). However, in other poeciliids (P. gracilis, G. falcatus, and G. metallicus), such elements were detected neither by PCR nor by Southern blot (fig. 6A ). Although Poecilia and Xiphophorus are sister groups (Rosen and Bailey 1963Citation ), the sequences isolated from Xiphophorus are closer to those of G. affinis and P. amates than to that of P. mexicana, suggesting the presence of two sublineages within lineage 2. As determined by quantitative slot-blotting (not shown), and according to figure 6A, lineage 2 underwent several major bursts of retrotransposition. This increased the copy number per haploid genome from less than 10 (G. affinis, P. mexicana, P. latipinna, and P. formosa) to about 200 for Xiphophorus (X. maculatus, X. helleri, X. nezahualcoyotl, and X. montezumae). After longer exposure of the Southern blot filters, faint signals were observed in Fundulus and in O. latipes, suggesting the presence of lineage 2 elements or cross-hybridization with other Rex1 variants. No signal was observed using the XimJ probe in more divergent fishes.

Lineage 3 was detected only in genome project databases from F. rubripes (FurB) and T. nigroviridis (Ten).

Lineage 4 contains sequences from O. latipes (Orl), B. baikalensis (Bab), A. anguilla (Ana), A. japonica (Anj), F. rubripes (FurA), C. labridens (Cil), O. niloticus (Orn), H. bimaculatus (Heb), and Fundulus (Fun) (fig. 7 ). In contrast to classical fish phylogeny (fig. 1 ), Fundulus elements were found to be more related to cichlid elements (Cil, Orn, and Fun) than to O. latipes sequences (Orl), suggesting the presence of additional sublineages in lineage 4.

Two distinct lineage 4 sublineages (Orl1 and Orl2-5) were identified in O. latipes. Orl1 is present at one copy per haploid genome (not shown), contains several stop codons disrupting the RT open reading frame, and is probably a "dead" element. Using Orl2 as a probe, the copy number of the Orl2-5 sublineage was estimated at about 30 copies per haploid genome (fig. 6C and data not shown). Using an Orn sequence as a probe, the copy number of Rex1 in O. niloticus was determined to be approximately 500 by quantitative slot-blotting (not shown). Using the Orn probe in Southern blot experiments, lineage 4 variant elements were detected at a high copy number in Fundulus but at less than 20 copies per haploid genome in all poeciliids tested (fig. 6D ). This showed the presence of a third Rex1 lineage in poeciliids in addition to lineages 1 and 2.

Related lineage 4 elements were detected in the European eel A. anguilla (Ana sequence) and in the Japanese eel A. japonica (Anj sequences) (fig. 7 ). Using Ana as a probe in Southern blot hybridization, lineage 4 elements were detected at low copy numbers (5 or less) in the genomes of both eels (fig. 6E ). They were also detected after filter overexposure using the Orl2 and Orn probes (data not shown). The high intensity of the smear in B. baikalensis DNA using the Ana element as a probe (fig. 6E ) indicates that Rex1 is highly reiterated in this fish. No signal was observed in A. sturio, C. carpio, D. rerio, E. lucius, or O. mykiss using the Orl, Orn, and Ana probes, confirming the results of the PCR analysis.

Age and Evolution of Rex1 Lineages
The phylogeny of Rex1 elements (fig. 7 ) shows that a Rex1 sequence is generally more related to sequences from the same fish than to sequences from another fish species. This shows the presence of numerous independent waves of retrotransposition having occurred from distinct master copies during genome evolution (see Deininger et al. 1992Citation ). At least 10 such major bursts of retrotransposition having occurred during fish evolution were detected (fig. 7 ). Some retrotransposition events are likely to be relatively recent, as revealed by the low Kimura-corrected average distance between sequences from lineage 2 within P. amates (4.5) and Xiphophorus (1.9–4.0) or between elements from lineage 4 within B. baikalensis (1.0) or O. niloticus (2.0) (table 2 ). Moreover, the rate of substitutions was higher at synonymous sites than at nonsynonymous sites in the great majority of interspecific comparisons (table 3 ). This probably reflects selection of master sequences with RT activity in Rex1 lineages and suggests frequent retrotransposition of Rex1 elements during fish evolution. On the other hand, the Ks/Ka ratio was closer to unity in comparisons between closely related sequences (table 3 ). This indicates that Rex1 elements are first influenced by pseudogene-like evolution after retrotransposition, as observed for other retroelements (McAllister and Werren 1997Citation ).


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Table 2 Kimura-Corrected Average Distances Between Rex1 Partial Reverse Transcriptase Genes

 

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Table 3 Average Ratios of Synonymous (Ks) to Nonsynonymous Substitutions (Ka) Between Rex1 Reverse Transcriptase Genes

 
Differences in the evolutionary rates of different elements can influence the topology of a phylogeny (Capy, Anxolabéhère, and Langin 1994Citation ; Cummings 1994Citation ). Analysis of the Kimura-corrected distance between Rex1 and Babar elements showed that Rex1 lineages evolved at roughly the same rate after divergence from their last common ancestor (table 2 ), with a possible exception represented by the dead element Orl1 from O. latipes. This was confirmed by analysis of the conceptual translation products (data not shown). Hence, we assume that the topology of the tree presented in figure 7 reflects the evolutionary history of Rex1 elements.

The distribution of Rex1 elements is discontinuous in lineage 4. Lineage 4 elements were detected in all Acanthopterygii (common ancestor in the main teleost lineage approximately 90 MYA; Benton 1990Citation ) and in both eels, A. anguilla and A. japonica (divergence from the main teleost lineage about 180 MYA). In contrast, no Rex1 element could be detected in C. carpio, D. rerio, E. lucius, and O. mykiss (divergence from the main teleost lineage 100–120 MYA). Ana and Anj elements are related and were detected reproducibly by PCR (tested for different organs in A. anguilla) and Southern blot hybridization in the genomes of both Anguilla species. Both Anguilla species were obtained from two different sources and were analyzed separately. Rex1 was never detected either in Acipenser DNA prepared and analyzed in parallel with A. anguilla DNA or in C. carpio, D. rerio, E. lucius, and O. mykiss DNA. Hence, a contamination problem could be ruled out.

The distribution of lineage 4 can be explained by two alternative hypotheses. The first hypothesis proposes that lineage 4 elements were present in the last common ancestor of all teleosts included in this study and have been transmitted vertically, i.e., from generation to generation. This would mean that evolutionary node G (fig. 7 ), leading to B. baikalensis and Anguilla elements, is at least 180 Myr old and that nodes A, E, and F are even older, indicating the presence of numerous Rex1 lineages before the separation of Anguilla from the main teleost lineage 180 MYA. Comparison of Bab with both Ana and Anj sequences (table 2 ) allowed us to calculate a very rough estimation of the rate of evolution of Rex1 elements if they really diverged 180 MYA: approximately 12% nucleotide substitution per 100 Myr. Using this value, nodes F and A would be at least approximately 250 and 450 Myr old, respectively, and the Rex1 lineage would have diverged from the Babar lineage at least 650 MYA, i.e., before the separation of the fish lineage from the major animal lineage. A complex evolutionary scenario is then necessary to explain the sporadic distribution of lineage 4: all Rex1 lineages would have been lost or would have rapidly diverged repeatedly and specifically in C. carpio, D. rerio, E. lucius, and O. mykiss lineages but would have been maintained in the Acanthopterygii.

In the second hypothesis, we consider the possibility of horizontal transfer (for reviews, see Kidwell 1993Citation ; Capy, Anxolabéhère, and Langin 1994Citation ; Cummings 1994Citation ). Horizontal transfer has been well documented for some DNA transposons and, more recently, for LTR retrotransposons (Gonzalez and Lessios 1999Citation ; Jordan, Matyunina, and McDonald 1999Citation ) but convincing evidence concerning non-LTR retrotransposons is much sparser (Kordis and Gubensek 1998Citation ; see Malik, Burke, and Eickbush 1999Citation for discussion). Non-LTR retrotransposons such as L1 are assumed to be (almost) exclusively transmitted vertically and can be used for phylogenetic analysis (Furano and Usdin 1995Citation ).

If we assume that node A is approximately 90–120 Myr old and arose after the separation of E. lucius and O. mykiss from the main teleost lineage but before the separation between Percomorpha and Cyprinodontiformes (see fig. 1 ), one event of horizontal transfer into a common ancestor of A. anguilla and A. japonica can simply explain the distribution of lineage 4 elements. Hence, horizontal transfer of Rex1 elements in the Anguilla genome is the most parsimonious explanation for the discontinuous distribution of lineage 4. The percentage of synonymous substitutions (Ks) between Anguilla and B. bakailensis Rex1 RT genes is lower than that between most other genes from Anguilla and Percomorpha (not shown). However, as other fish retrotransposons like the Rex3 LINE element evolved with a surprisingly low evolutionary rate (unpublished data), this observation cannot be used unambiguously to argue for horizontal transfer. Interestingly, none of the four partial Anguilla element sequences presented a corrupt partial RT open reading frame (table 1 ). Ana and Anj elements are present at a very low copy numbers in Anguilla genomes (fig. 6E ), indicating that they were not very active. One might expect that parasitic sequences should accumulate corrupting mutations when they are present for at least 180 Myr in a genome without transposing. Although corrupting mutations are likely to be present in other regions of the Anguilla elements, this observation suggests that Rex1 elements have been introduced into Anguilla genomes more recently. The Rex1 element found in Anguilla may hence represent one of the very rare examples of horizontal transfer of non-LTR retrotransposons in living organisms.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
We thank G. Schneider, H. Schwind, and P. Weber for fish maintenance, S. Chen, Y. Hong, and K. Mody (Würzburg, Germany), S. Kirilchik and M. Grachev (Irkutsk, Russia), and Y. Nakatsuru and T. Ishikawa (Tokyo, Japan) for fish organs and DNA, K. T. Cuong and A. K. Meyer for sequencing, J. Altenbuchner (Stuttgart, Germany) for the transposon system, C. Winkler for reading of the manuscript, and the Fugu and Tetraodon genome projects for making sequences available. This work was supported by grants to M.S. from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 465: Entwicklung und Manipulation pluripotenter Zellen), the European Commission (FAIR Project PL 97-3796, "Basis of sex determination and gonadal sex differentiation for sex control in aquaculture"), and the Fonds der Chemischen Industrie.


    Footnotes
 
Thomas Eickbush, Reviewing Editor

1 Abbreviations: AP, apurinic/apyrimidinic; LTR, long terminal repeat; nt, nucleotides; RT, reverse transcriptase. Back

2 Keywords: non-LTR retrotransposon reverse transcriptase phylogeny Xiphophorus teleost evolution Back

3 Address for correspondence and reprints: Jean-Nicolas Volff, Physiological Chemistry I, Biocenter, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany. E-mail: volff{at}biozentrum.uni-wuerzburg.de Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 

    Benton, M. J. 1990. Vertebrate palaeontology, biology and evolution. Unwin Hyman, London.

    Britten, R. J., T. J. McCormack, T. L. Mears, and E. H. Davidson. 1995. Gypsy/Ty3-class retrotransposons integrated in the DNA of herring, tunicate, and echinoderms. J. Mol. Evol. 40:13–24.[ISI][Medline]

    Burgtorf, C., K. Welzel, R. Hasenbank, G. Zehetner, S. Weis, and H. Lehrach. 1998. Gridded genomic libraries of different chordate species: a reference library system for basic and comparative genetic studies of chordate genomes. Genomics 52:230–232.

    Capy, P., D. Anxolabéhère, and T. Langin. 1994. The strange phylogenies of transposable elements: are horizontal transfers the only explanation? Trends Genet. 10:7–12.

    Cummings, M. P. 1994. Transmission patterns of eukaryotic transposable elements: arguments for and against horizontal transfer. Trends Ecol. Evol. 9:141–145.[ISI]

    Deininger, P. L., M. A. Batzer, C. A. Hutchinson, and M. H. Edgell. 1992. Master genes in mammalian repetitive DNA amplification. Trends Genet. 8:307–311.[ISI][Medline]

    Duvernell, D. D., and B. J. Turner. 1998. Swimmer 1, a new low-copy-number LINE family in teleost genomes with sequence similarity to mammalian L1. Mol. Biol. Evol. 15:1791–1793.[Free Full Text]

    ———. 1999. Variation and divergence of Death Valley pupfish populations at retrotransposon-defined loci. Mol. Biol. Evol. 16:363–371.[Free Full Text]

    Elgar, G., R. Sandford, S. Aparicio, A. Macrae, B. Venkatesh, and B. Brenner. 1996. Small is beautiful: comparative genomics with the pufferfish (Fugu rubripes). Trends Genet. 12:145–150.[ISI][Medline]

    Fischer, J., H. Maier, P. Viell, and J. Altenbuchner. 1996. The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66. Gene 180:81–89.

    Flavell, A. J., and D. B. Smith. 1992. A Ty1-copia group retrotransposon sequence in a vertebrate. Mol. Gen. Genet. 233:322–326.[ISI][Medline]

    Furano, A. V., and K. Usdin. 1995. DNA "fossils" and phylogenetic analysis. Using L1(LINE-1, long interspersed repeated) DNA to determine the evolutionary history of mammals. J. Biol. Chem. 270:25301–25304.[Free Full Text]

    Gonzalez, P., and H. A. Lessios. 1999. Evolution of sea urchin retroviral-like (SURL) elements: evidence from 40 echinoid species. Mol. Biol. Evol. 16:938–952.[Abstract]

    Hamada, M., N. Takasaki, J. D. Reist, A. L. DeCicco, A. Goto, and N. Okada. 1998. Detection of the ongoing sorting of ancestrally polymorphic SINEs toward fixation or loss in populations of two species of char during speciation. Genetics 150:301–311.

    Izsvák, Z., Z. Ivics, D. Garcia-Estefania, D. Fahrenkrug, and P. B. Hackett. 1996. DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio). Proc. Natl. Acad. Sci. USA 93:1077–1081.

    Izsvák, Z., Z. Ivics, and P. B. Hackett. 1995. Characterization of a Tcl-like transposable element in zebrafish (Danio rerio). Mol. Gen. Genet. 247:312–322.[ISI][Medline]

    ———. 1997. Repetitive elements and their genetic applications in zebrafish. Biochem. Cell Biol. 75:507–523.[ISI][Medline]

    Jordan, I. K., L. V. Matyunina, and J. F. McDonald. 1999. Evidence for the recent horizontal transfer of long terminal repeat retrotransposon. Proc. Natl. Acad. Sci. USA 96:12621–12625.

    Kallman, K. D. 1984. A new look at sex determination in poeciliid fishes. Pp. 95–171 in B. Turner, ed. Evolutionary genetics of fishes. Plenum, New York.

    Kazazian, H. H., and J. V. Moran. 1998. The impact of L1 retrotransposons on the human genome. Nat. Genet. 19:19–24.[ISI][Medline]

    Kidwell, M. G. 1993. Lateral transfer in natural populations of eukaryotes. Annu. Rev. Genet. 27:235–256.[ISI][Medline]

    Kidwell, M. G., and D. Lisch. 1997. Transposable elements as sources of variation in animals and plants. Proc. Natl. Acad. Sci. USA 94:7704–7711.

    Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111–120.[ISI][Medline]

    Koga, A., M. Suzuki, H. Inagaki, Y. Bessho, and H. Hori. 1996. Transposon element in fish. Nature 383:30.

    Kordis, D., and F. Gubensek. 1998. Unusual horizontal transfer of a long interspersed nuclear element between distant vertebrate classes. Proc. Natl. Acad. Sci. USA 95:10704–10709.

    Li, W. H. 1993. Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J. Mol. Evol. 36:96–99.[ISI][Medline]

    Li, W. H., C. I. Wu, and C. C. Luo. 1985. A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Mol. Biol. Evol. 2:150–174.[Abstract]

    McAllister, B. F., and J. H. Werren. 1997. Phylogenetic analysis of a retrotransposon with implication for strong evolutionary constraints on reverse transcriptase. Mol. Biol. Evol. 14:69–80.

    Malik, H. S., W. D. Burke, and T. H. Eickbush. 1999. The age and evolution of non-LTR-retrotransposable elements. Mol. Biol. Evol. 16:793–805.[Abstract]

    Meyer, A., and C. Lydeard. 1993. The evolution of copulatory organs, internal fertilization, placentae and viviparity in killifishes (Cyprinodontiformes) inferred from a DNA phylogeny of the tyrosine kinase gene X-src. Proc. R. Soc. Lond. B Biol. Sci. 254:153–162.

    Miller, K., C. Lynch, J. Martin, E. Herniou, and M. Tristem. 1999. Identification of multiple Gypsy LTR-retrotransposon lineages in vertebrate genomes. J. Mol. Evol. 49:358–366.[ISI][Medline]

    Ogiwara, I., M. Miya, K. Ohshima, and N. Okada. 1999. Retropositional parasitism of SINEs on LINEs: identification of SINEs and LINEs in elasmobranchs. Mol. Biol. Evol. 16:1238–1250.[Abstract]

    Ohshima, K., M. Hamada, Y. Teral, and N. Okada. 1996. The 3' ends of tRNA-derived short interspersed repetitive elements are derived from the 3' ends of long interspersed repetitive elements. Mol. Cell. Biol 16:3756–3764.

    Okada, N., M. Hamada, I. Ogiwara, and K. Ohshima. 1997. SINEs and LINEs share common 3' sequences: a review. Gene 205:229–243.

    Pamilo, P., and N. O. Bianchi. 1993. Evolution of the Zfx and Zfy genes: rates and interdependence between the genes. Mol. Biol. Evol. 10:271–281.[Abstract]

    Poulter, R., and M. Butler. 1998. A retrotransposon family from the pufferfish (fugu) Fugu rubripes. Gene 215:241–249.

    Poulter, R., M. Butler, and J. Ormandy. 1999. A LINE element from the pufferfish (fugu) Fugu rubripes which shows similarity to the CR1 family of non-LTR retrotransposons. Gene 227:169–179.

    Rosen, D. E., and R. M. Bailey. 1963. The poeciliid fishes (Cyprinodontiformes), their structure, zoogeography, and systematics. Bull. Am. Mus. Natl. Hist. 126:1–176.

    Ryan, M. J., and W. E. Wagner Jr. 1987. Asymmetries in mating preferences between species: female swordtails prefer heterospecific mates. Science 236:595–597.

    Schartl, M. 1995. Platyfish and swordtails: a genetic system for the analysis of molecular mechanisms in tumor formation. Trends Genet. 11:185–189.[ISI][Medline]

    Schartl, M., U. Hornung, H. Gutbrod, J.-N. Volff, and J. Wittbrodt. 1999. Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element. Genetics 153:1385–1394.

    Schartl, M., B. Wilde, I. Schlupp, and J. Parzefall. 1996. Evolutionary origin of a parthenoform, the amazon molly P. formosa, on the basis of molecular genealogy. Evolution 49:827–835.

    Swofford, D. L. 1989. PAUP: phylogenetic analysis using parsimony. Smithsonian Institution, Washington, D.C.

    Tristem, M., P. Kabat, E. Herniou, A. Karpas, and F. Hill. 1995. Ease1, a gypsy LTR-retrotransposon in the Salmonidae. Mol. Gen. Genet. 15:249–236.

    Venkatesh, B., and S. Brenner. 1997. Genomic structure and sequence of the pufferfish (Fugu rubripes) growth hormone-encoding gene: a comparative analysis of teleost growth hormone genes. Gene 187:211–215.

    Volff, J.-N., K. Körting, K. Sweeney, and M. Schartl. 1999. The non-LTR retrotransposon Rex3 from the fish Xiphophorus is widespread among teleosts. Mol. Biol. Evol. 16:1427–1438.[Abstract]

    Winkfein, R. J., R. D. Moir, S. A. Krawetz, J. Blanco, J. C. States, and G. H. Dixon. 1988. A new family of repetitive, retroposon-like sequences in the genome of rainbow trout. Eur. J. Biochem. 176:255–264.[Abstract]

Accepted for publication July 7, 2000.