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The Non-LTR Retrotransposon Rex3 from the Fish Xiphophorus is Widespread Among Teleosts Jean-Nicolas Volff, Cornelia Körting, Kimberley Sweeney,1 and Manfred Schartl Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg, Germany Rex3, the first reverse transcriptase (RT)-encoding retrotransposon isolated from the melanoma fish model Xiphophorus, is a non-long-terminal-repeat element related to the RTE family. The essential features of Rex3 are (1) an endonuclease and a reverse transcriptase, (2) 59 truncations of most of the copies, (3) a 39 tail consisting of tandem repeats of the sequence GATG, and (4) short target site sequence duplications of variable length. Compilation of Rex3 sequences from the pufferfish genome project suggested that, as observed for other members of the RTE family, no additional large open reading frame was present upstream of the endonuclease/reverse transcriptase open reading frame. There are about a thousand copies of Rex3 in the haploid genome of Xiphophorus, some of them probably resulting from recent retrotransposition events. Rex3 RNA was detected by RT-PCR in melanoma and in nontumorous tissues, as well as in melanoma-derived and embryonic cell lines. Rex3 is present in a broad panel of teleost species and was found in the promoter region and in introns of various genes. To our knowledge, Rex3 is the first autonomous retrotransposon described to date which is widespread in teleosts. This wide distribution and occasional association with coding sequences may confer on Rex3 a predisposition to play a role in genome evolution in teleosts. Introduction Fishes make up more than half of the 48,000 species of living vertebrates. They should therefore possess genetic tools for speciation-associated genome evolution. Transposons may be one of the factors fulfilling this function due to their ability to move within genomes, to generate mutations, and to influence genomic organization and gene expression in cells destined for the next generation (for review, see Kidwell and Lisch 1997; Kazazian and Moran 1998). There are two major classes of transposable elements. Class II transposons move as DNA molecules into another site of the host genome. In contrast, class I retroelements transpose via an RNA intermediate. Complete retrotransposons encode a reverse transcriptase copying the RNA molecule into DNA. Some retrotransposons, such as the gypsy element from Drosophila melanogaster, are flanked by long direct repeats that are involved in the transposition mechanism. They are closely related to retroviruses (Xiong and Eickbush 1990). Non-long-terminal-repeat (non-LTR) retrotransposons, also called long interspersed elements (LINEs), are frequently truncated at their 59 ends, probably because of incomplete reverse transcription of the RNA molecule. Line1, CR1, and Rte1 are the three major non-LTR retrotransposon lineages described for vertebrates (Haas et al. 1997; Kajikawa, Ohshima, and Okada 1997; Malik and Eickbush 1998). The retrotransposon-encoded reverse transcrip1 Present address: Max Planck Institute für Experimentelle Endokrinologie, Hannover, Germany. Abbreviations: aa, amino acids; EN, apurinic-apyrimidic endonuclease; LTR, long terminal repeat; nt, nucleotides; RT, reverse transcriptase; UTR, untranslated region. Key words: non-LTR retrotransposon, RTE family, reverse transcriptase, phylogeny, Xiphophorus, teleost evolution. Address for correspondence and reprints: Jean-Nicolas Volff, Physiological Chemistry I, Biocenter, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany. E-mail: [email protected]. Mol. Biol. Evol. 16(11):1427–1438. 1999 q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 tase (RT) may be involved in the mobilization of nonautonomous sequences such as short interspersed elements (SINEs) and retro(pseudo)genes. The 39 ends of tRNA-derived SINEs are related to the 39 ends of LINE elements (Ohshima et al. 1996; Okada et al. 1997). Finally, miniature inverted-repeat transposable elements (MITEs) form a third group of mobile elements whose transposition mechanism is not yet understood. All groups of transposable elements are represented in fish (Izsvák, Ivics, and Hackett 1997). Examples include the DNA transposons Tol2 and Sleeping Beauty from the medakafish Oryzias latipes and the zebrafish Danio rerio, respectively (Izsvák, Ivics, and Hackett 1995; Koga et al. 1996), the SINE family DANA (Izsvák et al. 1996), or the MITE Angel (Izsvák, Ivics, and Hackett 1997). LTR retrotransposons are present in herring and some members of the Salmonidae (Flavell and Smith 1992; Britten et al. 1995; Tristem et al. 1995). A full-length LTR retrotransposon has been reported from the pufferfish Fugu rubripes (Poulter and Butler 1998) using sequences from the Fugu genome project (Elgar et al. 1996). Non-LTR retrotransposon partial sequences have been reported from the genomes of rainbow trout (Winkfein et al. 1988), eel (Ohshima et al. 1996), and zebrafish (Okada et al. 1997). A Line1-related retrotransposon called Swimmer 1 (SW1) has been found in the medakafish Oryzias latipes and in the desert pupfish Cyprinodon macularius (Duvernell and Turner 1998). Maui, an element showing similarity to those of the CR1 family of non-LTR retrotransposons, is present in the genome of F. rubripes (Poulter, Butler, and Ormandy 1999). To our knowledge, an RT-carrying retrotransposon has not previously been reported as being widespread in bony fishes or even in teleosts. Fish of the genus Xiphophorus (Poeciliidae) are inhabitants of freshwater biotopes of the Atlantic drainage of Mexico, Honduras, and Guatemala. Formation of spontaneous melanoma in certain hybrid genotypes of Xiphophorus is caused by a dominant genetic locus, designated Tu (for review, see Schartl 1995). In feral pa1427 1428 Volff et al. rental fish, no tumor occurs due to the presence of a tumor-modifier locus (R) that suppresses Tu action. Elimination of R-bearing chromosomes by successive crossings with another Xiphophorus species having neither Tu nor R allows expression of the oncogenic potential of Tu in some hybrids. Molecular cloning revealed that the Tu locus includes the Xmrk oncogene, which encodes a novel receptor tyrosine kinase (Wittbrodt et al. 1989). The Tu locus emerged through nonhomologous recombination of the Xmrk proto-oncogene with a previously uncharacterized sequence called D. This event generated an additional copy of Xmrk with a new promoter. Suppression of the new Xmrk promoter by R in parental fish and its deregulation in hybrids explains the genetics of melanoma formation in Xiphophorus (Schartl 1995). With about 750 Mb (Tiersch et al. 1989; Vinogradov 1998; D. Lamatsch, personal communication), the Xiphophorus haploid genome is almost twice the size of the ‘‘compact’’ genome of the sequence project fish F. rubripes (Elgar et al. 1996) and is about four times as small as the human genome. Xiphophorus genomic DNA contains approximately 90% single-copy sequences, with the remaining 10% being predominantly multiple-copy elements (Schwab 1982). Except for the D locus, which is repeated as 20–50 copies per haploid genome (Förnzler et al. 1996), and the XIR sequence, which may correspond to a retroviral LTR (Anders et al. 1994), almost nothing is known about repetitive DNA in Xiphophorus. We have begun to construct a cosmid contig from the Xmrk oncogene/proto-oncogene region of the Xiphophorus sex chromosomes. In one of the cosmids from the Y chromosome, we identified several repetitive elements. One of them, Rex3, is the first RT-encoding retrotransposon to be found in Xiphophorus and the first example of a retrotransposon being widespread in teleosts. Materials and Methods Fish and Cell Lines The following fish, maintained under standard conditions (Kallman 1975) and kept as randomly inbred lines at the Biocenter of Würzburg, were used (strain designation or geographical origin in brackets): Xiphophorus maculatus (Rio Jamapa, Rio Usumacinta), Xiphophorus milleri (Laguna Catemaco), Xiphophorus helleri (Rio Lantecilla), Xiphophorus couchianus (Apodaca), Xiphophorus malinche (Rio Calnali), Xiphophorus meyeri (Musquiz), Xiphophorus nezahualcoyotl (Rio El Salto), Xiphophorus montezumae (Cascadas de Tamasopo), Xiphophorus variatus (Ciudad Mante), Xiphophorus cortezi (Rio Axtla), Gambusia affinis affinis (Pena Blanca), Poeciliopsis gracilis (Rio Jamapa), Heterandria bimaculata (Tierra Blanca), Heterandria formosa (Fort Lauderdale), Phallichtys amates (aquarium stock), Poecilia mexicana (Media Luna), Poecilia latipinna (Key Largo), Poecilia formosa (Tampico), Girardinus metallicus (aquarium stock), Girardinus falcatus (aquarium stock), Fundulus sp. (Laguna de Labradores), O. latipes (medakafish strains HB32c and Car- bio), D. rerio (zebrafish strain m14), and Cichlasoma labridens (Cascadas de Tamasopo). Common carp (Cyprinus carpio), European eel (Anguilla anguilla), and sturgeon (Acipenser sturio) were obtained from a local fish farm. Genomic DNA from Nile tilapia (Oreochromis niloticus) and from Chinese perch (Siniperca chuatsi) were a gift from S. Chen (University of Würzburg). Genomic DNA and organs from Battrachocottus baikalensis (Baikal Lake) were kindly provided by S. Kirilchik and M. Grachev (Institute of Limnology, Irkutsk, Russia). Hemichromis bimaculatus (Comoe National Park, Ivory Coast) was a gift from K. Mody (University of Würzburg). A2 is an embryonal cell line from Xiphophorus xiphidium (Kuhn, Vielkind, and Anders 1979), and PSM is a cell line derived from a melanoma from an X. maculatus/X. helleri hybrid (Wakamatsu et al. 1981). SdSr 24, XhIII, and Sd/hIIIBCn are embryonic cell lines from X. maculatus, X. helleri, and X. maculatus/X. helleri backcross hybrids, respectively (M. Pagany, University of Würzburg, personal communication). Cloning, Sequencing, and Southern Blot Analysis Fish genomic DNA was isolated as described (Schartl et al. 1996). The genomic library of X. maculatus used in this study consists of 35–45-kb inserts cloned into cosmid Lawrist7 (Burgtorf et al. 1998). All PCR reactions were done in 5% DMSO with an annealing temperature of 558C. PCR products were cloned into plasmid pUC18 using the SureClone ligation kit (Amersham Pharmacia Biotech). To generate a probe for Southern blot hybridization, a 1,142-bp fragment containing the whole RT-encoding sequence of Rex3 was amplified with primers RTX3-F1 (59-TACGGAGAAAACCCATTTCG-39) and RTX3-R1 (59-AAAGTTCCTCGGTGGCAAGG-39) and cloned into pUC18, giving rise to plasmid pROST30. For estimation of intercopy sequence variability, sequence-encoding domains 1, 2, 2A, A, and B of the RT were amplified by PCR from genomic DNA from one X. maculatus Rio Jamapa, one X. maculatus Rio Usumacinta, and one X. helleri using primers RTX3-F3 (59-CGGTGAYAAAGGGCAGCCCTG) and RTX3-R3 (59-TGGCAGACNGGGGTGGTGGT). PCR products were sequenced on both strands. Sequencing of larger fragments was achieved using a transposon mutagenesis system (Fischer et al. 1996). Sequencing reactions were performed using the ThermoSequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech) and run on ALF and ALFexpress automated laser fluorescent sequencers (Amersham Pharmacia Biotech). For Southern blot analysis, genomic DNA was blotted after digestion with restriction enzymes onto positively charged nylon membranes and hybridized with the insert of pROST30 at 428C in 35% formamide, 0.1% Na-pyrophosphate, 50 mM Tris-Cl (pH 7.5), 5 3 SSC, 1% sodium dodecyl sulfate (SDS), 5 3 Denhardt’s solution, and 100 mg/ml calf thymus DNA. Filters were washed with 2 3 SSC–1% SDS at 508C (low stringency) or with 0.1 3 SSC–1% SDS at 688C (high stringency). Rex3 copy number estimation was done by quan- Rex3 Retrotransposon in Teleosts titative slot-blotting. After DNA-DNA hybridization with the insert of pROST30, the intensity of 5 mg and 500 ng of EcoRI-digested genomic DNA (both blotted in duplicate) was compared to that of blotted serial dilutions of known amounts of pROST30. Before blotting the pROST30 reference, the plasmid was cut with restriction enzymes and mixed with either 5 mg or 500 ng of human placenta DNA to mimic the presence of nonhybridizing DNA in the fish genomic DNA. Nucleotide Sequence Analysis Nucleotide sequences were analyzed using programs of the GCG Wisconsin package, version 9.1 (Genetics Computer Group [GCG], Madison, Wis.). Multiple-sequence alignments were generated using PileUp of GCG. Sequence similarity searches were done using BLAST 2.0 (Altschul et al. 1997) on the server of the National Center for Biotechnology Information (http:// www4.ncbi.nlm.nih.gov/BLAST). Gene structure analysis was performed with the Nucleotide Identify X (NIX) system on the UK-HGMP Resource Centre server (http://menu.hgmp.mrc.ac.uk/menu-bin/Nix/Nix.pl). Protein sequence motifs were searched using programs contained in MOTIF (http://www.motif.genome.ad.jp) from the GenomeNet server (Universities of Kyoto and Tokyo). Phylogenetic analyses were done with PAUP (Swofford 1989) as part of the GCG package. Heuristic and branch-and-bound tree searches were performed using both parsimony and distance (minimum evolution). Neighbor-joining trees were also constructed. Robustness of the trees was tested by bootstrap analyses (100 replicates). Expression Analysis Organs originating from different individual fish with the same genotype were pooled and prepared using the TRIZOL reagent (GIBCO Life Technologies). Reverse transcription reactions were performed with 200 ng total RNA per reaction. PCR amplification was done as described above using primers RTX3-F2 (59-AACACCTTGGCTGCGCCTAG-39) and RTX3-R1. The 650-bp fragment amplified with these primers contains the sequence encoding domains 0, 1, 2, 2A, A, and B of the RT. Accession Numbers Sequences of Rex3a-XmJ, Rex3b-XmJ, and Rex3cXmJ are deposited in GenBank under accession numbers AF125981, AF125982, and AF125983, respectively. The sequence of Rex3-Fr is available on request. Accession numbers for other sequences used in this study are from GenBank/EMBL unless otherwise noted. Results Genomic Organization of Rex3 in X. maculatus Using a genomic library (Burgtorf et al. 1998), we are currently constructing a cosmid contig of the Xmrk oncogene/proto-oncogene-containing region of the sex chromosome of X. maculatus. Sequencing of cosmid D18 097, whose insert covers approximately 45 kb of the Y chromosome, revealed the presence of three new 1429 RT-carrying retrotransposons, which were called Rex1, Rex2, and Rex3 (retroelement of Xiphophorus 1, 2, and 3). Rex1 and Rex2 are non-LTR retrotransposons. Rex2 is closely related to the Maui element from F. rubripes (Poulter, Butler, and Ormandy 1999) while Rex1 could not be assigned to the known major families of nonLTR retrotransposons (unpublished data). A fragment from Rex3 was amplified by PCR using primers RTX3-F1 and RTX3-R1, cloned, and used as a probe in Southern blot hybridization experiments to identify additional Rex3 copies in other cosmids which were previously isolated. Two copies of Rex3 were found in cosmids M05 080 and B17 126, which were originally isolated with a DNA probe from the D locus (unpublished data). The Rex3 copies from cosmids D18 097, M05 080, and B17 126 were called Rex3a-XmJ, Rex3b-XmJ, and Rex3c-XmJ, respectively (XmJ for Xiphophorus maculatus, population Rio Jamapa; fig. 1). The lengths of Rex3a-XmJ, Rex3b-XmJ, and Rex3c-XmJ are 2,109, 2,624, and 2,223 nt, respectively. They all have RT domains; however, partial apurinic-apyrimidic endonuclease (EN) domains could be detected only in Rex3b-XmJ and Rex3c-XmJ (see below). No large conserved LTR flanking Rex3 could be found, indicating that this element belongs to the family of non-LTR retrotransposons. The 39 end of Rex3 consists of two GAA repeats followed by GATG tandem repeats (between 8 and 17, depending on the copy). The stop codon that terminates the C-terminal-domain-encoding sequence is located at a distance of only 3 nt upstream of the first GAA repeat. This indicates that the 39 untranslated region (UTR) of Rex3 is extremely short. Small target sequence duplications of various lengths (13, 10, and 6 nt) were observed. Two of them presented a common core of 6 bp (CTGATG) containing the sequence GATG identical to that of the 39 tandem repeats, and the third one contained the sequence ATG (G was missing compared with GATG). This suggests that integration of Rex3 does not occur totally randomly but preferentially at sites with similarity to the repeats constituting the 39 tail of the element. Two CpG islands were predicted in Rex3 (predicted as ‘‘good’’ and ‘‘excellent’’, respectively, by using the GRAIL/CpG program as part of the NIX package). Rex3a-XmJ contains a palindromic sequence not detected in either of the other copies. Inspection of the sequences indicated the presence of an inversion in Rex3a-XmJ compared with Rex3b-XmJ and Rex3cXmJ. The left repeat of this palindrome is in fact the start of the inversion. Rex3 encodes an RT with the nine conserved domains found in non-LTR retrotransposons (fig. 2; Xiong and Eickbush 1990; Malik and Eickbush 1998). We identified an additional 17-aa domain which was also present in the other non-LTR retrotransposons analyzed. This domain, which was called F, presents a conserved tyrosine residue and other amino acids occurring very frequently at several positions. A conserved leucine residue was found downstream of this domain followed by two charged amino acids (mostly RR; F9 in fig. 2). In contrast, the F domain was not 1430 Volff et al. FIG. 1.—Structures of copies of Rex3 in Xiphophorus maculatus (Rex3-XmJ), Oryzias latipes (Rex3-Ol), and Fugu rubripes (Rex3-Fr). Sequence duplications, possibly generated during integration, are boxed. Overlined sequences are the palindromic sequence present in Rex3aXmJ and the corresponding sequence in Rex3b-XmJ and Rex3c-XmJ. ‘‘A→B’’ indicates the region inverted in Rex3a-XmJ compared with the other Xiphophorus copies. The four sequences shown under Rex3-Fr are the four genome survey sequences from the Fugu genome project, which presumably include the 59 end of full-length Rex3 elements. The arrow indicates the nucleotide which has been arbitrarily chosen as the first Rex3 nucleotide. The region of similarity between the four sequences is shaded at the positions at which at least three nucleotides are identical. Two predicted CpG islands are shown. The sequence flanking the Ct domain on its 39 side are the next nucleotides following the stop codon. Abbreviations: EN, endonuclease; RT, reverse transcriptase; Ct, C-terminal domain. The accession number for Rex3-Ol is AB021490. obvious, for example, in the LTR retrotransposon gypsy from D. melanogaster (SwissProt accession number P10401). Rex3 RT has the greatest similarity with the RTs of the Rte1 elements from Caenorhabditis elegans (Yougman, van Luenen, and Plasterk 1996; accession number AF054983) and SR2 from Schistosoma mansoni (accession number AF025672). Rex3b-XmJ RT displays 41% similarity (30.7% identity) with Rte1 and 39.5% similarity (32% identity) with SR2. Using a partial sequence of SR2, Malik and Eickbush (1998) classified SR2 and Rte1 in the same class of non-LTR retrotransposons, which they called RTE. Phylogenetic analyses of the RT domains of different non-LTR retrotransposons confirmed that Rex3 is most closely related to the RTE class (fig. 3). A partial EN domain was detected in Rex3b-XmJ and Rex3c-XmJ (fig. 4). In Rex3, the serine of the highly conserved sequence SD was replaced by a threonine. In the most complete copy of Rex3b-XmJ, between 120 and 240 aa of the EN N-terminus are missing compared with other non-LTR retroelements. This means that all three copies are truncated at their 59 ends, which is not unusual for non-LTR retrotransposons. The lengths of the intervening domains located between EN and RT were 210 and 173 aa plus one and two gaps due to the presence of stop codons, respectively, for Rex3b-XmJ and Rex3c-XmJ. This is similar to the size of this domain in Rte1 (185 aa), SR2 (200 aa), or L1H (Line1 from human, 210 aa). The size difference between Rex3b-XmJ and Rex3c-XmJ is probably due to one or two deletion events in Rex3c-XmJ having also removed the last amino acids of the C-terminal part of the endonuclease. Only the 49 last amino acids of the intervening domain directly flanking the RT domain were found in Rex3a-XmJ. The intervening domain of Rex3b-XmJ has similarity with the corresponding domains of SR2 (48 aa, 35% identity) and of LINE-like elements from Neurospora crassa (61 aa, 22% identity, PIR accession number 43275) and from the snail Biomphalaria glabrata (58 aa, 25% identity, PIR accession number PC1123). The Rex3 C-terminal domain shows some similarity to those of Rte1 and SR2 and to that of Art2, another member from the RTE family which was initially identified as a SINE. A potential AT-hook DNA-binding site was detected (fig. 5). AT-hook is a small motif which has a typical sequence pattern centered around a glycine-arginine-proline (GRP) tripeptide which is necessary and sufficient to bind DNA (Aravind and Landsman 1998). Although a threonine or a methionine was found instead of the preferred residues at position 9, threonines and methionines have been found in several other examples of AT-hook motifs described in ftp: //ncbi.nlm.nih.gov/pub/landsman/hmg-i/classI, /classII and /classIII. No RNase H domain could be detected. Rex3 Retrotransposon in Teleosts 1431 FIG. 2.—Comparison of the reverse transcriptase domain of Rex3 with that of other non-LTR retrotransposons. The conserved domains (0 to E) with their conserved amino-acids ‘‘|’’ are essentially the same as those described by Xiong and Eickbush (1990) and Malik and Eickbush (1998). Possible conservation in non-LTR retrotransposons of one additional domain (F) and of a leucine residue followed by two charged residues (F9) is shown in open boxes. Amino acids frequently found in F and F9 are indicated by ‘‘1.’’ Included in this analysis are (accession numbers in parentheses): Doc from Drosophila melanogater (PIR S13329), Jockey from D. melanogaster (SwissProt P21328), Juan from Aedes aegypti (M95171), CR1 from Gallus gallus (U88211), Q from Anopheles gambiae (U03849), Rex3-Ol from Oryzias latipes (AB021490), SR2 from Schistosoma mansoni (AF025672), Rte1 from Caenorhabditis elegans (AF054983), human Line-1 (L1H, PIR B28096), and SW1 from Oryzias latipes (AF055640). 1432 Volff et al. FIG. 3.—Positioning of Rex3 into non-LTR retrotransposon phylogeny. Phylogenetic analysis was done with the reverse transcriptase domains shown in figure 2. The 50% majority-rule consensus tree shown here has been rooted on the SW1/L1H clade according to Malik, Burke, and Eickbush (1999). Minimal and maximal bootstrap values obtained using the different types of analysis (100 replicates, see Materials and Methods) are given. Copy Number and Intercopy Sequence Variability in Xiphophorus Using the insert of pROST30 as a probe in quantitative slot blot analysis (see Materials and Methods), the copy number of Rex3 was found to be almost constant in the genus Xiphophorus and was estimated to be around 1,000 per haploid genome for X. maculatus Rio Jamapa and Rio Usumacinta, X. milleri, X. helleri, X. couchianus, X. nezahualcoyotl, X. montezumae, X. variatus, and X. cortezi. Intercopy variability was assessed by sequencing a 421-bp sequence belonging to the RT domain, which was amplified by PCR. Thirteen, eleven, and five indi- vidual PCR products were sequenced for X. maculatus Rio Jamapa (RJ), X. maculatus Rio Usumacinta (RU), and X. helleri, respectively (sequences available on request). The intraindividuum intercopy variability was between 0.5% and 8.8% for RJ, between 0.2% and 3.6% for RU, and between 0.7% and 8.6% for X. helleri. The interpopulation (RJ/RU) variability was between 0% and 9.5% (one copy of RJ identical to one copy of RU). Interspecific variability ranged from 0.2% to 10.0% between X. maculatus and X. helleri. Phylogenetic analysis indicated that the most recent common ancestor of all of these copies was present before the divergence of the X. maculatus and X. helleri species (data not shown). Comparison of the whole sequences of Rex3a-XmJ, Rex3b-XmJ, and Rex3c-XmJ revealed an intercopy variability comprising between 3.7% and 8.4%. These values are compatible with those reported for the 421-bp fragment. This indicates that the PCR method has probably not biased the estimation of the intercopy variability. Of the 29 PCR products sequenced, only 4 had mutations disrupting the partial RT open reading frame present in the 421-nt fragment. Detection of Transcripts Containing Rex3 Sequences in Xiphophorus RNA containing sequences corresponding to the Nterminal part of Rex3 RT were detected by RT-PCR in benign and malignant tumors, as well as in healthy tissues from hybrid fish with melanoma (fig. 6). This was not specific for hybrid genotypes, because the same result was obtained for the liver of purebred healthy X. maculatus. The same picture was obtained using cell lines: Rex3 RNA was detected in all cell lines tested, independent of their origins (melanoma-derived or embryonal, parental or hybrid genotype). In Silico Reconstruction of a Rex3 Element from the Pufferfish F. rubripes Searching the database of the pufferfish F. rubripes genome project (Elgar et al. 1996; http://fugu.hgmp.mrc. FIG. 4.—Comparison of the partial apurinic-apyrimidic endonuclease domain of Rex3 with those of other LTR retrotransposons. Conserved residues (according to Malik and Eickbush [1998] and references therein) are indicated by ‘‘|’’ for identical and ‘‘1’’ for similar amino acids. The DDBJ accession number for BCNT from Bos taurus is AB005652. All other accession numbers are given in the legend to figure 1. Rex3 Retrotransposon in Teleosts 1433 FIG. 5.—Comparison of the C-terminal domain of Rex3 with that of several members of the RTE family. Identical and similar residues are indicated by ‘‘|’’ and ‘‘1,’’ respectively. Amino acids compatible with an AT-hook DNA-binding site are shaded in the multiple-sequence alignment. Preferred residues (k, p, r, and/or g) at the corresponding positions of the core sequence of the AT-hook motifs are indicated in the ‘‘AT-hook’’ shaded box. The nucleotide consensus sequence of the Art2 element from Muntiacus muntjak is found under accession number X82879. All other accession numbers are given in the legend to figure 1. ac.uk) revealed more than 100 genome survey sequences (GSS) matching significantly with Rex3b-XmJ over its complete length (E values between 1e-115 and 4e10). By compilation of 73 GSSs (each GSS is approximately 300–700 nt in length), we were able to construct an artificial sequence of the pufferfish Rex3 element that FIG. 6.—RT-PCR detection of Rex3 RNA in different fish tissues and cell lines. The 650-bp fragment detected contains the sequence encoding domains 0 to B of the reverse transcriptase (see Materials and Methods). 1 RT 5 plus reverse transcriptase; 2 RT 5 without reverse transcriptase, demonstrating the absence of contaminating genomic DNA. we called Rex3-Fr. Particularly, the sequence of Rex3 could be extended into the 59 direction compared with the Xiphophorus copies. The Rex3-Fr sequence is 3,348 nt in length (sequence available on request) and displays 86.5% identity with the whole nucleotide sequence of Rex3b-XmJ. As observed for the Xiphophorus copies, Rex3-Fr encodes an RT and ends with GATG tandem repeats (figs. 1 and 2). The deduced amino acid sequence of the endonuclease shows the conserved amino acid residues present in other complete endonucleases, except for a glutamic acid residue which was replaced by a serine (fig. 4). Three in-frame ATG codons are located upstream of the endonuclease domain (fig. 4). Interestingly, on the 59 side of the sequence contig assembled to determine the Rex3-Fr sequence, the similarity between the four last GSSs ends approximately at the same position on the 59 side (fig. 1). No significant similarity between the four sequences could be found upstream of this breakpoint. Such a situation is not expected in the case of copies presenting random 59 truncations. This strongly suggests that the end-of-homology region indeed corresponds to the 59 ends of the fulllength Rex3 elements of the pufferfish. The start of the Rex3 element was assigned arbitrarily to the nucleotide shown in figure 1. Nevertheless, sequencing of several complete genomic copies and determination of their target site sequence duplications are necessary to confirm this assumption. While 18 GSSs of the pufferfish database contain the 39 end of Rex3, only four sequences include the 59 end. This may indicate that most of the Rex3 copies are truncated at their 59 ends in F. rubripes, as observed for X. maculatus. Assuming that Rex3-Fr represents the full-length sequence, the size of the 59 UTR is between 157 and 316 nt, depending on the ATG used as start codon. Interestingly, no large open reading 1434 Volff et al. FIG. 7.—Southern blot analysis of the distribution of Rex3 in bony fish. Genomic DNA was cut with EcoRI. Filters were hybridized with the insert of pROST30, which contains the whole reverse-transcriptase-encoding sequence of Rex3, and washed under low stringency (2 3 SSC, 508C). Filters were exposed at 2808C with intensifying screen for 12 h (A) and 3 days (B). Signals obtained with sturgeon DNA were not significantly stronger than the presumably nonspecific signals obtained with human DNA. After washing with higher stringency (0.1 3 SSC, 688C), signals were considerably weaker for eel. frame preceding the EN/RT open reading frame could be found in Rex3. Such an open reading frame is also absent from the C. elegans Rte1 element but is detected in all other non-LTR retrotransposons (Malik and Eickbush 1998). This suggests that lack of the first large open reading frame may be a characteristic of Rte1-related retrotransposons. Unlike Rte1 (Malik and Eickbush 1998), Rex3 has no small open reading frame partially overlapping the EN/RT-encoding sequence on its 59 side. Distribution of Rex3 in Bony Fishes Using the insert of pROST30 as a probe in Southern blot hybridization, Rex3 was detected in all members of Poeciliidae and in the closely related Fundulus, as well as in other teleosts including eel (A. anguilla), carp (C. carpio), zebrafish (D. rerio), cichlids from different geographical origins, Chinese perch (S. chuatsi) and medakafish (O. latipes) (fig. 7 and table 1). In contrast, no significant signal was detected for the sturgeon (A. sturio). All these results were confirmed by PCR analysis using nine different combinations of primers matching the Rex3 RT domain (data not shown). This means either that Rex3 is absent from the sturgeon or that the sequence divergence was too high to allow for Rex3 detection. By searching sequence databases, several Rex3-related sequences were found, including a sequence tagged site (STS number Z13870) from the genetic linkage map of the zebrafish (http://zebrafish.mgh.harvard. edu; E 5 1e-41). Another Rex3-related sequence from the RT domain was found in intron 6 of the Y allele of the ONC-Xmrk oncogene of Xiphophorus (accession number AF092693). A truncated copy of Rex3 is also integrated into one intron of the membrane-bound guanylyl cyclase B gene of the medakafish (N. Suzuki, Hokkaido University, personal communication; DDBJ accession number AB021490). This copy, which we called Rex3-Ol, shows 82%–83% identity to the Xiphophorus copies at the DNA level and contains only a part of the RT domain (fig. 1). Rex3-Ol ends with six GATG repeats and is flanked by an 11-nt sequence duplication, possibly generated during integration. This duplication is not directly adjacent to the GATG repeats and does not contain the ATG sequence found in the three integration sites of Xiphophorus. A truncated copy of Rex3 lacking the 59 end of the EN domain is present in the partial sequence of the F. rubripes immunoglobulin heavy-chain gene cluster (accession number AF108422). In the same fish, three copies of Rex3 were found at the T-cell receptor alpha locus (13.8 kb, accession number AF110526). Two of these copies show both 59 and 39 ends as defined in Rex3-Fr but have a 2-kb internal deletion removing the 39 part of the EN and almost all of the RT domain. The third copy is a truncated element lacking the EN-encoding region and about one half of the RT domain. A sequence matching the 39 end of Rex3 was found in the promoter of the antifreeze protein (AFP) gene from the ocean pout Macrozoarces americanus (Du, Gong, and Hew 1992). The sequence similarity includes two GATG repeats. The Rex3-related region is located approximately 2 kb upstream of the TATA box. The region of similarity may extend 59 from Rex3 Retrotransposon in Teleosts 1435 Table 1 Distribution of Rex3 in Bony Fish Fish (Osteichthyes–Actinopterygii) Rex3 Chondrostei . . . . . . . . . . . . Acipensiformes Acipenseridae Acipenser sturio 2 Neopterygii–Teleostei Elopomorpha . . . . . . . . . Anguilliformes Anguillidae Anguilla anguilla 1 Euteleostei Ostariophysi . . . . . . . . Cypriniformes Cyprinidae Perciformes Cichlidae Tetraodontiformes Scorpaeniformes Cyprinodontiformes Serranidae Zoarcidae Tetraodontidae Cottidae Poeciliidae Cyprinus carpio Danio rerioa Oreochromis niloticus Hemichromis bimaculatus Cichlasoma labridens Siniperca chuatsi Macrozoarces americanusb Fugu rubripes c Battrachocottus baikalensis Xiphophorus maculatus Xiphophorus milleri Xiphophorus helleri Xiphophorus couchianus Xiphophorus malinche Xiphophorus meyeri Xiphophorus nezahualcoyotl Xiphophorus montezumae Xiphophorus variatus Xiphophorus cortezi Gambusia affinis Poeciliopsis gracilis Heterandria bimaculata Heterandria formosa Phallichytes amates Poecilia mexicana Poecilia latipinna Poecilia formosa Girardinus metallicus Girardinus falcatus Fundulus sp. Oryzias latipesd 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Acanthopterygii . . . . . Fundulidae Adrianichthyidae NOTE.—The presence (1) or absence (2) of Rex3 was assessed by Southern blot hybridization unless otherwise indicated. a Assessed by Southern blot and sequence tagged site. b Assessed by antifreeze protein gene promoter. c Assessed by genome survey sequences, immunoglobulin heavy-chain gene cluster, T-cell receptor alpha locus, and HSP70-2 gene promoter. d Assessed by Southern blot and intron of the guanylyl cyclase B gene. the published ocean pout sequence. Another Rex3-related sequence was found approximately 700 nt from the ATG codon of the heat shock protein HSP70-2 gene of F. rubripes (accession number Y08577). Similarity to Rex3 also included five GATG repeats. The sequence directly flanking the Rex3-related region on its 59 side shows no similarity with Rex3 and is found in the F. rubripes HSP70-3 gene (accession number Y08578), suggesting that the region of similarity to Rex3 may not extend in the 59 direction. The Rex3-related region was not found in the HSP70-3 sequence. Discussion We identified and characterized a new non-LTR retrotransposon from the genome of the fish Xiphophorus. Sequencing of several copies of Rex3 revealed that complete versions of this element should encode an endonuclease and a reverse transcriptase. The three Xiphophorus copies sequenced so far, as well as other sequences present in databases, are truncated at their 59 ends, probably due to an incomplete reverse transcrip- tion of the RNA intermediate. This phenomenon is often observed for non-LTR retrotransposons but seems to be very rare for the Line1-related SW1 element from medakafish (Duvernell and Turner 1998). Sequence comparison of Rex3 with other non-LTR retrotransposons uncovered an additional putative conserved region downstream of the nine RT domains described previously (Malik and Eickbush 1998). The RT region of non-LTR retrotransposons has been independently expanded by another group as well (Malik, Burke, and Eickbush 1999). High conservation of primary structure is suggestive of an important role in enzyme three-dimensional structure and/or biochemical activity. Although the function of this domain is still unclear, we assigned it to the RT domain. This may be consistent with the observation that the region required for RT activity is significantly larger than just the highly conserved regions originally defined by Xiong and Eickbush (1990) (Martin et al. 1998). Comparative analysis of the reverse transcriptase, endonuclease, and C-terminal domains indicated that 1436 Volff et al. Rex3 is closely related to the RTE class of retroelements. Although members of this class have been found in the genomes of organisms as different as nematodes, molluscs, insects, and mammals (Malik and Eickbush 1998), Rex3 is, to our knowledge, the first RTE family-related retroelement to be described in the fish lineage. Rex3 shares structural features with members of the RTE group, including (1) the absence of flanking large direct repeats, (2) variable-length target site duplication, (3) the presence of large 59 truncations in most of the copies, (4) a very short 39 untranslated region, and (5) a 39 tail consisting of oligonucleotide tandem repeats. Moreover, the Rex3 sequence obtained by compilation of sequences from the F. rubripes genome project showed no large open reading frame upstream of the EN/RTencoding region. Such an open reading frame, which may encode an RNA-binding protein, is found in most of the non-LTR retrotransposons but not in the Rte1 element of C. elegans (Yougman, van Luenen, and Plasterk 1996; Malik and Eickbush 1998). If the sequence from F. rubripes is representative for complete elements, the sizes of full-length Rex3 and Rte1 elements are very similar (approximately 3.3 kb). Hence, the structural features of Rex3 corroborate the phylogenetic analysis of its RT and place Rex3 into the RTE family of non-LTR retrotransposons. The oligonucleotide sequence (GATG)n of the 39 tail of Rex3 is different from those reported to date for RTE-like elements. The GATG tail seems to be a signature of Rex3, because it has been found in the three Xiphophorus elements as well as in the Rex3 sequences found in the databases. It has been suggested that such 39 tandem repeats may prime reverse transcription by hybridization to homologous sequences at nicked chromosomal sites (Burch, Davis, and Haas 1993). The presence in the Xiphophorus copies of Rex3 of the GATG sequence in two target site duplications and of the ATG sequence in the third one suggests that similarity between 39-tail and target sequence increases the probability of insertion. Nevertheless, a 3–4-nt identity is not necessary for integration, because neither the GATG nor the ATG motif could be found in the potential sequence duplication generated by insertion of the medakafish element. As observed for Rte1, no cysteine/histidine-rich domain was found in the C-terminal part of the EN-RT protein. This domain, whose role remains obscure (maybe binding to DNA), is needed for efficient transposition of Line1 (Moran et al. 1996). We detected a potential AT-hook DNA-binding domain in Rex3 and in some members of the RTE class. This DNA-binding motif may play a role in the transposition mechanism. Such a domain was detected in the transposase of the Tcl family of class II transposons (Aravind and Landsman 1998), but we could not identify such a motif in Line1, Jockey, or CR1 retrotransposons. Rex3 retrotransposition should require a full-length sense strand RNA as a transposition intermediate. Because of their pronounced 59 truncation and/or the presence of stop codons in the region encoding the intervening domain, the copies isolated from Xiphophorus are probably not capable of autonomous retrotransposition. As suggested by the almost perfect identity between some partial sequences, some events of Rex3 retrotransposition may have occurred very recently. This could be explained either by the presence of Rex3 autonomous elements in the genome of Xiphophorus or by the presence of other autonomous retrotransposons (such as Rex1 and Rex2, for example), which may provide the enzymatic facilities required for jumping of Rex3-truncated elements. Autonomous Rex3 elements, if they exist, may also play a role in the mobilization of other nonautonomous elements such as the SINE DANA, originally found in zebrafish (Izsvák et al. 1996) but also present in Xiphophorus (unpublished data), or the Xiphophorus TX-1 element, which exhibits features of a retrotransposon but does not encode any of the enzymes that are required for its mobilization (unpublished data). Rex3 is, to our knowledge, the first retrotransposon reported to be widespread in teleosts. Some Rex3 copies are associated with coding regions. Examples of Rex3 insertions into introns and into/at the proximity of the promoter regions of genes are documented in this paper. Rex3 RNA, which was detected in all tissues and cell lines tested, may be the result of the presence of Rex3 in transcription units, but may also arise, alternatively and probably more rarely, from full- length autonomous elements. Association of Rex3 with coding regions may be an important factor for genome evolution in teleosts via modification of the expression level or specificity of neighboring genes. There is a growing list of examples of transposon insertions providing benefits to the host (Britten 1997; Kidwell and Lisch 1997). Transposons can, of course, directly inactivate genes by insertion into their coding regions or can serve as a substrate for DNA recombination due to their repeated nature. This can induce deletions, amplifications, inversions, or translocations which may participate in the evolution of a genome. Inversions and translocations can also place a gene into a new genomic context and consequently modify its expression. Finally, it remains to be determined whether Rex3 could be at the origin of SINEs, as observed for members of the RTE class (Malik and Eickbush 1998) and for other retrotransposons (Ohshima et al. 1996; Okada et al. 1997). One example of a Rex3derived SINE may be the short sequence found upstream of the F. rubripes HSP70-2. Nevertheless, we could not find any Pol III promoter upstream of this element, indicating that this sequence is probably an extremely truncated Rex3 element rather than an authentic SINE. Acknowledgments We are grateful to G. Schneider, H. Schwind, and P. Weber for breeding and maintenance of fish, to S. Chen, Y. Hong, U. Hornung, K. Mody, M. Pagany, and C. Winkler (University of Würzburg, Germany) and S. Kirilchik and M. Grachev (Institute of Limnology, Irkutsk, Russia) for the gift of fish organs, DNA, and RNA, to K. T. Cuong for DNA sequencing, and to Y. Hong for helping to prepare fish for DNA isolation. Many thanks to J. Altenbuchner (University of Stuttgart, Rex3 Retrotransposon in Teleosts Germany) for helpful discussions and for providing us the transposon mutagenesis system and to N. Suzuki (Hokkaido University, Japan) for allowing us to use the medakafish Rex3 sequence. We are grateful to J. Altschmied and C. Winkler for critical reading of the manuscript and to the team of the Fugu genome project for making the genome survey sequences available to the public. This work was supported by grants to M.S. from the Deutsche Forschungsgemeinschaft through 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. LITERATURE CITED ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHÄFFER, J. ZHANG, Z. ZHANG, W. MILLER, and D. J. LIPMAN. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. ANDERS, A., H. PETRY, C. FLEMING, K. PETRY, P. BRIX, W. LÜKE, H. GRÖGER, E. SCHNEIDER, J. KIEFER, and F. ANDERS. 1994. Increasing melanoma incidence: putatively explainable by retrotransposons. Experimental contributions of the Xiphophorine Gordon-Kosswig Melanoma system. Pigment Cell Res. 7:433–450. ARAVIND, L., and D. LANDSMAN. 1998. AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res. 26:4413–4421. BRITTEN, R. J. 1997. Mobile elements inserted in the distant past have taken on important functions. Gene 205:177–182. 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. BURCH, J. B. E., D. L. DAVIS, and N. B. HAAS. 1993. Chicken repeat 1 elements contain a pol-like open reading frame and belong to the non-long terminal repeat class of retrotransposons. Proc. Natl. Acad. Sci. USA 90:8199–8203. 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. DU, S. J., Z. GONG, and C. L. HEW. 1992. Development of an all-fish gene cassette for gene transfer in aquaculture. Mol. Mar. Biol. Biotechnol. 1:290–300. 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. 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. 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. FÖRNZLER, D., J. ALTSCHMIED, I. NANDA, R. KOLB, M. BAUDLER, M. SCHMID, and M. SCHARTL. 1996. The Xmrk onco- 1437 gene promoter is derived from a novel amplified locus of unusual organisation. Genome Res. 6:102–113. HAAS, N. B., J. M. GRABOWSKI, A. B. SIVITZ, and J. B. BURCH. 1997. Chicken repeat 1 (CR1) elements, which define an ancient family of vertebrate non-LTR retrotransposons, contains two closely spaced open reading frames. Gene 197: 305–309. 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. . 1997. Repetitive elements and their genetic applications in zebrafish. Biochem. Cell Biol. 75:507–523. KAJIKAWA, M., K. OHSHIMA, and N. OKADA. 1997. Determination of the entire sequence of turtle CR1: the first open reading frame of the turtle CR1 element encodes a protein with a novel zinc finger motif. Mol. Biol. Evol. 14:1206– 1217. KALLMAN, K. D. 1975. The platyfish, Xiphophorus maculatus. Pp. 81–132 in R. C. KING, ed. Handbook of genetics. Plenum Press, New York. KAZAZIAN, H. H., and J. V. MORAN. 1998. The impact of L1 retrotransposons on the human genome. Nat. Genet. 19:19– 24. 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. KOGA, A., M. SUZUKI, H. INAGAKI, Y. BESSHO, and H. HORI. 1996. Transposon element in fish. Nature 383:30. KUHN, C., U. VIELKIND, and F. ANDERS. 1979. Cell cultures derived from embryos and melanoma of poeciliid fish. In Vitro 15:537–544. 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. MALIK, H. S., and T. H. EICKBUSH. 1998. The RTE class of non-LTR retrotransposons is widely distributed in animals and is the origin of many SINEs. Mol. Biol. Evol. 15:1123– 1134. MARTIN, S. L., J. LI, L. E. EPPERSON, and B. LIEBERMAN. 1998. Functional reverse transcriptase encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis. Gene 215:69–75. MORAN, J. V., S. E. HOLMES, T. P. NAAS, R. J. DEBERARDINIS, J. D. BOEKE, and H. H. KAZAZIAN. 1996. High frequency retrotransposition in cultured mammalian cells. Cell 87: 917–927. OHSHIMA, K., M. HAMADA, Y. TERAL, and N. OKADA. 1996. The 39 ends of tRNA-derived short interspersed repetitive elements are derived from the 39 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 39 sequences: a review. Gene 205:229–243. 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. 1438 Volff et al. SCHARTL, M. 1995. Platyfish and swordtails: a genetic system for the analysis of molecular mechanisms in tumor formation. Trends Genet. 11:185–189. 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. SCHWAB, M. 1982. Genome organisation in Xiphophorus (Poeciliidae; Teleostei). Mol. Gen. Genet. 188:410–417. SWOFFORD, D. L. 1989. PAUP: phylogenetic analysis using parsimony. Version 9.1. Smithsonian Institution, Washington, D.C. TIERSCH, T. R., R. W. CHANDLER, K. D. KALLMAN, and S. S. WACHTEL. 1989. Estimation of nuclear DNA content by flow cytometry in fishes of the genus Xiphophorus. Comp. Biochem. Physiol. 94B:465–468. 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. VINOGRADOV, A. E. 1998. Genome size and GC-percent in vertebrates as determined by flow cytometry: the triangular relationship. Cytometry 31:100–109. WAKAMATSU, Y. 1981. Establishment of a cell line from the platyfish-swordtail hybrid melanoma. Cancer Res. 41:679– 680. 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. WITTBRODT, J., D. ADAM, B. MALITSCHEK, W. MÄUELER, F. RAULF, A. TELLING, S. M. ROBERTSON, and M. SCHARTL. 1989. Novel putative receptor kinase encoded by the melanoma-inducing Tu locus in Xiphophorus. Nature 341:415– 421. XIONG, Y., and T. H. EICKBUSH. 1990. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9:3353–3362. YOUGMAN, S., H. G. A. M. VAN LUENEN, and R. H. A. PLASTERK. 1996. Rte-1, a retrotransposon-like element in Caenorhabditis elegans. FEBS Lett. 380:1–7. THOMAS EICKBUSH, reviewing editor Accepted July 5, 1999