Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg, Germany
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Abstract |
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Introduction |
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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. 1996
; Schartl et al. 1999
). Several evolutionary studies have already been performed on fish retroelements (e.g., Hamada et al. 1998
; Duvernell and Turner 1999
), 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 1984
; Ryan and Wagner 1987
; Meyer and Lydeard 1993
; Schartl 1995
). We describe here the non-LTR retrotransposon Rex1 from the platyfish Xiphophorus maculatus and its complex evolutionary dynamics in teleost genomes.
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Materials and Methods |
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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)
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)
and Pamilo and Bianchi (1993)
, and applies Kimura's (1980)
two-parameter method. Phylogenetic analyses were done with PAUP* (Swofford 1989
) 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 AJ288431AJ288486. 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.
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Results and Discussion |
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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 1999
). 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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Comparison of Rex1 untranslated regions revealed a segment with a high degree of conservation in the 3' terminal region about 200300 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. 1999
), suggests that this region may serve as a recognition site for Rex1-encoded reverse transcriptase.
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Multiple Rex1 Lineages in Teleosts
PCR amplification of the sequence encoding RT domains 37 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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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 1963
), 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. 1992
). 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 (1.94.0) 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 1997
).
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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 1990
) 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 100120 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 1993
; Capy, Anxolabéhère, and Langin 1994
; Cummings 1994
). Horizontal transfer has been well documented for some DNA transposons and, more recently, for LTR retrotransposons (Gonzalez and Lessios 1999
; Jordan, Matyunina, and McDonald 1999
) but convincing evidence concerning non-LTR retrotransposons is much sparser (Kordis and Gubensek 1998
; see Malik, Burke, and Eickbush 1999
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 1995
).
If we assume that node A is approximately 90120 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.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: AP, apurinic/apyrimidinic; LTR, long terminal repeat; nt, nucleotides; RT, reverse transcriptase.
2 Keywords: non-LTR retrotransposon
reverse transcriptase
phylogeny
Xiphophorus
teleost evolution
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
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