Jule from the Fish Xiphophorus Is the First Complete Vertebrate Ty3/Gypsy Retrotransposon from the Mag Family

Jean-Nicolas Volff, Cornelia Körting, Joachim Altschmied, Jutta Duschl, Kimberley Sweeney, Katrin Wichert, Alexander Froschauer and Manfred Schartl

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


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Jule is the second complete long-terminal-repeat (LTR) Ty3/Gypsy retrotransposon identified to date in vertebrates. Jule, first isolated from the poeciliid fish Xiphophorus maculatus, is 4.8 kb in length, is flanked by two 202-bp LTRs, and encodes Gag (structural core protein) and Pol (protease, reverse transcriptase, RNase H, and integrase, in that order) but no envelope. There are three to four copies of Jule per haploid genome in X. maculatus. Two of them are located in a subtelomeric region of the sex chromosomes, where they are associated with the Xmrk receptor tyrosine kinase genes, of which oncogenic versions are responsible for the formation of hereditary melanoma in Xiphophorus. One almost intact copy of Jule was found in the first intron of the X-chromosomal allele of the Xmrk proto-oncogene, and a second, more corrupted copy is present only 56 nt downstream of the polyadenylation signal of the Xmrk oncogene. Jule-related elements were detected by Southern blot hybridization with less than 10 copies per haploid genome in numerous other poeciliids, as well as in more divergent fishes, including the medakafish Oryzias latipes and the tilapia Oreochromis niloticus. Database searches also identified Jule-related sequences in the zebrafish Danio rerio and in both genome project pufferfishes, Fugu rubripes and Tetraodon nigroviridis. Phylogenetic analysis revealed that Jule is the first member of the Mag family of Ty3/Gypsy retrotransposons described to date in vertebrates. This family includes the silkworm Mag and sea urchin SURL retrotransposons, as well as sequences from the nematode Caenorhabditis elegans. Additional related elements were identified in the genomes of the malaria mosquito Anopheles gambiae and the nematode Ascaris lumbricoides. Phylogeny of Mag-related elements suggested that the Mag family of retrotransposons is polyphyletic and is constituted of several ancient lineages that diverged before their host genomes more than 600 MYA.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Long-terminal-repeat (LTR) retrotransposons from the Ty3/Gypsy superfamily (metaviridae) are phylogenetically more related to vertebrate retroviruses (retroviridae) than to the second major superfamily of LTR retrotransposons, the Ty1/Copia class (pseudoviridae) (Xiong and Eickbush 1990Citation ). Besides LTRs and the genes gag (encoding the structural core protein) and pol (encoding protease, reverse transcriptase, RNase H, and integrase), some Ty3/Gypsy-related elements display another similarity to retroviruses: an envelope (env) gene, which might indicate their infectious nature (Pelisson et al. 1994Citation ; Lerat and Capy 1999Citation ; Pantazidis, Labrador, and Fontdevilla 1999Citation ; Peterson-Burch et al. 2000Citation ). Accordingly, the Gypsy retrotransposon from Drosophila melanogaster was shown to be infectious (Kim et al. 1994Citation ; Song et al. 1994, 1997Citation ). Some LTR retrotransposons from the Ty3/Gypsy superfamily have occasionally been transmitted horizontally: the envelope-encoding Gypsy element has jumped between species of the Drosophila melanogaster subgroup (Terzian et al. 2000), and the sea urchin retroviral-like SURL elements (no apparent envelope gene) have been transferred between echinoid species (Gonzalez and Lessios 1999Citation ). Phylogenetic analysis using the sum of the amino acids in the reverse transcriptase, RNase H, and integrase domains revealed different families within the Ty3/Gypsy superfamily (Malik and Eickbush 1999Citation ).

To our knowledge, only one complete Ty3/Gypsy-like retrotransposon has been isolated to date from vertebrates: the Sushi retrotransposon from the Japanese pufferfish Fugu rubripes (Poulter and Butler 1998Citation ). Sushi belongs to the Ty3 family and is related to Maggy from the rice blast fungus Magaporthe grisea (Farman et al. 1996Citation ; Poulter and Butler 1998Citation ; Malik and Eickbush 1999Citation ). Other partial elements (mostly partial pol sequences) have been identified in lampreys, fishes, amphibians, and reptiles (Britten et al. 1995Citation ; Tristem et al. 1995Citation ; Marracci et al. 1996Citation ; Miller et al. 1999Citation ). While Hsr1 from the terrestrial salamander Hydromantes displays a higher degree of similarity to the pufferfish Sushi element (Marracci et al. 1996Citation ; Poulter and Butler 1998Citation ; data not shown), the other vertebrate partial pol sequences are difficult to assign to a particular Ty3/Gypsy family because of their shortness and the lack of resolution afforded by the analyses of individual enzymatic domains (Malik and Eickbush 1999Citation and references therein).

The fish genus Xiphophorus (Teleostei: Poeciliidae) is an established model for cancer research and for the analysis of sex determination and many questions of evolutionary biology (Kallman 1984Citation ; Ryan and Wagner 1987Citation ; Meyer and Lydeard 1993Citation ; Schartl 1995Citation ). Overexpression of the receptor tyrosine kinase oncogene Xmrk is responsible for the formation of hereditary melanoma in Xiphophorus (for review, see Schartl 1995Citation ). The Xmrk oncogene has been formed by gene duplication of the preexisting Xmrk proto-oncogene. Both the Xmrk proto-oncogene and the oncogene are located in the subtelomeric region of the X and Y sex chromosomes in the platyfish Xiphophorus maculatus (strain Rio Jamapa), where they are closely linked to the sex determination locus (Gutbrod and Schartl 1999Citation ; Nanda et al. 2000Citation ; unpublished results). Several repetitive DNA elements, including a Y chromosome–specific large cluster of LTR-like sequences, the non-LTR retrotransposons Rex1 and Rex3, and the nonautonomous LTR element TX-1, have already been identified in this region (Schartl et al. 1999Citation ; Volff et al. 1999Citation ; Nanda et al. 2000Citation ; Volff, Körting, and Schartl 2000Citation ). We report here the discovery of Jule, a novel vertebrate LTR retrotransposon from the Ty3/Gypsy superfamily belonging to the Mag family. Although there are only three to four copies of Jule per haploid genome in X. maculatus, two of them are associated with the Xmrk genes on the sex chromosomes of the platyfish.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Fishes
The following fishes were from stocks maintained at the University of Würzburg: X. maculatus (Rio Jamapa, Rio Usumacinta), Xiphophorus milleri (Laguna Catemaco), Xiphophorus helleri (Rio Lantecilla), Poecilia mexicana (Media Luna), Gambusia affinis (Pena Blanca), Girardinus falcatus (aquarium stock), Poeciliopsis gracilis (Rio Jamapa), Fundulus sp. (Laguna de Labradores), Oryzias latipes (medakafish strain HB32c), Danio rerio (zebrafish strain m14), and Oreochromis niloticus (Rio Purification). 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. Genomic DNA and organs from Batrachocottus baikalensis were a gift from S. Kirilchik and M. Grachev (Institute of Limnology, Irkutsk, Russia).

DNA Manipulations
Genomic DNA was isolated as described (Schartl et al. 1996Citation ). The genomic library of X. maculatus consists of 35–45-kb inserts cloned into cosmid Lawrist7 (Burgtorf et al. 1998Citation ). Cosmid sequencing was performed by transposon mutagenesis (Fischer et al. 1996Citation ). Sequencing reactions were performed using the ThermoSequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech) and run on an ALFexpress automated laser fluorescent sequencer (Amersham Pharmacia Biotech). The DNA probe used in Southern blot hybridization experiments was a 1.8-kb fragment from the pol gene of Jule (fig. 1 ) cloned into plasmid pJOE2114 (Fischer et al. 1996Citation ), resulting in plasmid pROST56-214. This fragment was obtained by transposon mutagenesis of an original Jule 7.3-kb EcoRI fragment cloned into pJOE2114 (plasmid pROST56). The 3' end of the 1.8-kb fragment corresponded to the unique internal EcoRI restriction site in Jule located at approximately the C-terminal domain of the integrase (fig. 1 ). Because there is no other EcoRI site in Jule, this probe is likely to reveal different copies of Jule after cutting genomic DNA with EcoRI. For Southern blot analysis, genomic DNA was blotted after restriction enzyme digestion onto positively charged nylon membranes and hybridized with the insert of pROST56-214 at 42°C in 35% formamide, 0.1% Na-pyrophosphate, 50 mM Tris-Cl (pH 7.5), 5 x SSC, 1% sodium dodecyl sulfate (SDS), 5 x Denhardt's solution, and 100 µg/ml calf thymus DNA. Filters were washed with 2 x SSC–1% SDS at 50°C (low stringency).



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Fig. 1.—Comparison of the structure of two copies of the Jule retrotransposon of Xiphophorus maculatus with those of other retrotransposons from the Mag family. Black boxes represent the long terminal repeats (LTRs). White and black triangles show the position, of nonsense and frameshift mutations, respectively. Sequences of the terminal inverted repeats and the target site duplications are shown over the LTRs and on both sides of the retrotransposons, respectively. EN = endonuclease; Int = integrase; Prot = protease; RH = RNase H

 
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 of GCG. Phylogenetic analyses were performed with PAUP* (Swofford 1989Citation ) as part of the GCG package. Trees were generated 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
The nucleotide sequences reported in this paper have been deposited in the GenBank database under accession numbers AF278691 and AF278692. The sequence alignment used to generate the Ty3/Gypsy retrotransposon phylogeny has been deposited in the EMBL Nucleotide Sequence Database under accession number DS43387.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
A New LTR Retrotransposon on the Sex Chromosomes of the Platyfish X. maculatus
Using a gridded genomic library of X. maculatus strain Rio Jamapa (Burgtorf et al. 1998Citation ), we are currently constructing and analyzing a cosmid contig covering the Xmrk proto-oncogene/oncogene region of both the X and the Y sex chromosomes of the platyfish. Using Southern blot hybridization, gene/allele-specific PCR reactions, and DNA sequencing, the X and Y chromosome alleles of the Xmrk proto-oncogene and oncogene were identified in some cosmids (unpublished data). Cosmid G17 069 contains the 5' flanking genomic region, exon 1, and the majority of intron 1 of the X allele of the Xmrk proto-oncogene (data not shown). Cosmid GO8 133 includes the major part of the coding region of the Y allele of the Xmrk oncogene (the 3' part) and the 3' flanking genomic region (Nanda et al. 2000).

A 4.8-kb sequence delimited by two 202-nt LTRs and overlapping two 6.5- and 7.5-kb genomic EcoRI fragments was detected in cosmid G17 069 (accession number AF278691). The conceptual translation product of this sequence shows a significant degree of similarity to several LTR retrotransposon–encoded proteins (see below). Hence, this 4.8-kb sequence probably corresponds to a new fish LTR retrotransposon, which we called Jule-1 (for Jumping long element; fig. 1 ). A second, longer Jule element (9.2 kb, accession number AF278691) was detected in cosmid GO8 133 (Jule-2; fig. 1 ). The 9.2-kb sequence includes a 7.3-kb EcoRI fragment and ends in two 2.0- and 6.3-kb genomic EcoRI fragments.

Structural Analysis of Jule Elements
Jule encodes a putative translation product with the traditional LTR retrotransposon domains Gag (structural core protein) and Pol (protease, reverse transcriptase, RNase H, and integrase, in that order) (figs. 1 and 2 ). No Env (envelope) domain or gene was detected. Jule Gag protein contains two putative CCHC nucleic acid–binding sites: a canonical CX2CX4HX4C zinc finger, already described in retroviruses and several other retrotransposons, preceded by a slightly different CX2CX3HX4C motif, which has been found in some other retrotransposons (Springer, Davidson, and Britten 1991Citation ; Pardue et al. 1996Citation ). As observed for other integrases, Jule integrase contains an HH-CC zinc finger–like domain in its N-terminal domain, along with the classical DD35E motif (fig. 2 ). Neither the conserved GPY/F domain nor the putative chromodomain (Malik and Eickbush 1999Citation ) could be detected in the integrase of Jule. The reverse trancriptase of Jule contains all classical conserved domains according to Xiong and Eickbush (1990)Citation (not shown).



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Fig. 2.—Comparison of Jule and Mag retrotransposon protein sequences. Gag potential zinc fingers and reverse transcriptase and integrase domains are overlined; the Pol protease active site and the RNase H conserved motif are underlined. Conserved residues in the HHCC and DD35E integrase domains are indicated by asterisks. Identical residues are shown in black, and conservative substitutions shown in gray (drawn using MacBoxshade). Abbreviations are as in figure 1 . Accession numbers are given in the legend of figure 4

 
The sequence of Jule-1 is compatible with the presence of a unique gag-pol open reading frame without frameshift between gag and pol. The gag-pol open reading frame of Jule-1 is disrupted only by a stop codon in the integrase domain (fig. 1 ). Nevertheless, examination of the degree of similarity to other integrases in this region suggested the presence of two successive frameshifts, one approximately 25 nt upstream of the stop codon, and the second about 40 nt downstream from the stop codon, restoring the original reading frame. Jule-2 is more corrupted than Jule-1 (fig. 1 ). Several frameshift and nonsense mutations interrupt the gag-pol open reading frame. Two sequences are also integrated in Jule-2. The non-LTR retrotransposon Rex5, a novel element related to Maui from the Japanese pufferfish Fugu rubripes (Poulter, Butler, and Ormandy 1998Citation ), is integrated in an opposite orientation (according to the sense of transcription) in the beginning of gag (interrupting the Gag protein after residues DTYGL; fig. 2 ). Furthermore, a 1.2-kb sequence of unknown origin is integrated within the CX2CX3HX4C motif-encoding sequence. Jule-2 also has a 350-nt deletion in the pol region (fig. 1 ). Jule-1 and Jule-2 appear to be extremely closely related; they display 97.7% nucleotide identity within the Jule sequences.

Jule is delimited by two 3-nt inverted repeats (TGT and ACA) corresponding to the extremities of the LTRs (fig. 1 ). A promoter was predicted between positions 131 and 181 of the LTRs, with a transcriptional starting site at position 174 (NNPP/eukaryotic promoter prediction, BCM Search Launcher; Smith et al. 1996Citation ). A potential polyadenylation signal ATTAAA was detected at position 140. The ATTAAA sequence displays an activity comparable to that of the canonical polyadenylation sequence AATAAA (Zhao, Hyman, and Moore 1999Citation ). Several LTR retrotransposons use cellular tRNAs as primers for reverse transcription (Mak and Kleiman 1997Citation ). No tRNA candidate could be unambiguously identified in the case of Jule. As observed for numerous other retrotransposons (Boeke and Chapman 1991Citation ), a polypurine tract (AAAAGGGGAGGAA) is adjacent to the 3' LTR. Jule elements are flanked by 5-nt direct repeats which probably correspond to the duplication of the target sequence generated during integration of the retrotransposon. The two Jule elements sequenced show different target duplication sequences: CTCTA for Jule-1, and GTCTT for Jule-2.

Association Between Jule Retrotransposons and Xmrk Genes
Jule-1 was identified in cosmid G17 069, which contains the 5' part of the X allele of the Xmrk proto-oncogene. Further sequencing showed that Jule-1 is in fact integrated in the first intron of this gene, 802 nt downstream from the first exon, in an opposite orientation to Xmrk. No Jule element was found at the same position in the Y allele of the Xmrk proto-oncogene or in both alleles of the Xmrk oncogene (not shown). Jule-2 was identified in cosmid GO8 133, which includes a large part of the 3' portion of the Y allele of the Xmrk oncogene and approximately 20 kb of 3' genomic flanking sequence. Jule-2 is integrated in the same orientation as Xmrk only 56 nt downstream of the polyadenylation signal of this gene. Using the insert of plasmid pROST56-214 (Jule-specific; see Materials and Methods) and a Rex5-specific probe in Southern blot hybridization experiments, Jule and Rex5 were both detected 3' of the X allele of the Xmrk oncogene as well (not shown). Nevertheless, the sizes and numbers of EcoRI fragments detected were different: the Jule and Rex5 probes hybridized with the same 7.3-kb fragment on the Y chromosome but with two different fragments on the X chromosome (4.8 and 6.1 kb, respectively). This suggests either the presence of X and/or Y chromosome-specific mutations after integration of both retrotransposons, or, alternatively, that they are not integrated exactly at the same position.

Jule Belongs to the Mag Family of Ty3/Gypsy Retrotransposons
Among sequences present in protein databases, the Gag-Pol polyprotein of Jule shows the highest similarity to proteins encoded by the retrotransposon Mag from the silkworm Bombyx mori (Michaille et al. 1990Citation ; Garel, Nony, and Prudhomme 1994Citation ; fig. 2 ). In particular, the Jule polyprotein displays 40% amino acid identity (425 of 1,054 residues) and 57% amino acid similarity (612 of 1,054 residues) to the Pol polyprotein of Mag (expected value E = 0.0). The Mag family of Ty3-Gypsy retrotransposons also includes the echinoid retrotransposon SURL from Tripneustes gratilla (sea urchin retroviral-like retrotransposon; Springer, Davidson, and Britten 1991Citation ) and several sequences from the nematode Caenorhabditis elegans (Malik and Eickbush 1999Citation ) (fig. 1 ). Phylogenetic analysis of the Pol protein of Mag-related C. elegans retrotransposons revealed the presence of different subgroups in the genome of this nematode. Nevertheless, all of these subgroups were included in a single C. elegans–specific group in a global phylogenetic tree of Mag-related retrotransposons (data not shown). Hence, only one subgroup, represented by an element located on C. elegans chromosome III, was used for further analysis (accession number Z38112; three mismatches were introduced to regenerate the amino acid sequence; figs. 1 and 3 ). Another copy of this element is also present on chromosome V (accession number ZZ99711), and solo LTR sequences were found on chromosomes II (AL110494), III (ALZ99293), IV (AL132862), and V (Z81489). This element is 5.1 kb in length, is delimited by two 218-nt LTRs, and is flanked by 5-nt target site duplications (CTAAA). Only one CX2CX4HX4C zinc finger was detected in the Gag protein of this C. elegans element.

We identified a corrupted Mag/Jule-related element, detected in a database genomic sequence from the nematode Ascaris lumbricoides (accession number L22247; fig. 3 ) and identified as containing the small nuclear RNA U2-1 gene and the U2-2 pseudogene (Shambaugh, Hannon, and Nilsen 1994Citation ). This element is truncated at its 3' end (the integrase-encoding region is absent; fig. 3 ) and corrupted by several frameshifts. The truncated element is flanked by two perfect 2.2-kb inverted repeats, each containing the U2-1 and U2-2 (pseudo)genes (not shown). Protease, reverse transcriptase, and RnaseH domains could be identified in the partial Pol sequence (fig. 3 and data not shown). The Gag sequence contains a CX2CX3PX4C-X6-CX2CX4HX4C motif reminiscent of the CX2CX3HX4C-X6-CX2CX4HX4C double zinc finger domain present in Jule. Searching public databases for other sequences similar to Mag and Jule led to the identification and partial reconstruction of two different retrotransposons from the African malaria mosquito Anopheles gambiae. These sequences were generated by the Genoscope sequencing center (www.genoscope.cns.fr). Partial reconstruction of reverse transcriptase/RNase H domains of A. gambiae retrotransposon 1 was done using sequences including AL152894, AL155210, and AL141680 (fig. 3 ). The Pol region from reverse transcriptase to integrase of A. gambiae retrotransposon 2 was obtained using sequences including AL139931, AL147383, and AL14105 (fig. 3 ). Because of the lack of sequences available, reconstruction could not be further extended to determine the complete structure of these elements.



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Fig. 3.—Sequence comparison of the reverse transcriptase, RNase H, and integrase domains of retrotransposons from the Mag family. Identical residues are shown in black, and conservative substitutions shown in gray (drawn using MacBoxshade). Conserved residues in the HHCC and DD35E integrase domains are indicated by asterisks. Abbreviations are as in figure 1 . Accession numbers are given in the legend of figure 4

 
Study of the phylogeny of Ty3/Gypsy retrotransposons has been restricted by the lack of resolution using individual enzymatic domains (Malik and Eickbush 1999Citation and references therein). To overcome this problem, we performed a phylogenetic analysis using the sum of the amino acid sequences of the reverse transcriptase, RNase H, and integrase domains according to Malik and Eickbush (1999)Citation (figs. 3 and 4 ). Although sequences of the A. gambiae retrotransposon 1 and the A. lumbricoides element are not complete, they were included in the study, because restricting analysis to the overlapping regions (from the start of the reverse transcriptase to the SRLPWNSPDN sequence in Jule; fig. 3 ) did not introduce significant differences into the topology of the tree (not shown). Two vertebrate retroviruses were used as outgroups according to Malik and Eickbush (1999)Citation .

Jule, Mag, SURL, and the A. gambiae, A. lumbricoides, and C. elegans elements were found to belong to the so-called Mag family of retrotransposons (fig. 4 ). To our knowledge, Jule is the first retrotransposon from the Mag family that has been identified to date in vertebrates. Jule is clearly more related to elements from the Mag family than to the other complete vertebrate Ty3/Gypsy retrotransposon Sushi from the pufferfish F. rubripes (Poulter and Butler 1998Citation ), which clearly belongs to the Ty3 family (fig. 4 ). Furthermore, we found no evidence for a closer relationship between Jule and the different partial Ty3/Gypsy sequences already isolated from vertebrates (see Introduction), which were too short to be included in the phylogenetic analysis.



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Fig. 4.—Phylogenetic analysis of the Mag family of Ty3/Gypsy retrotransposons. This analysis was performed using the sum of the amino acid sequences of the reverse transcriptase, RNase H, and integrase domains according to Malik and Eickbush (1999). Both human foamy virus and feline leukemia virus were chosen as outgroups for this 50% majority-rule consensus tree according to Malik and Eickbush (1999). Minimal and maximal bootstrap values obtained using the different kinds of analysis are shown (see Materials and Methods). The sequence alignment used has been deposited in the EMBL Nucleotide Sequence Database under accession number DS43387. Sequence accession numbers are as follows: Jule—consensus translation of AF278691 and AF278692; Anopheles gambiae retrotransposon 1—consensus translation of genome project sequences including AL152894, AL155210, and AL141680; Mag—S08405; SURL—M75723; A. gambiae retrotransposon 2—consensus translation of genome project sequences including AL139931, AL147383, and AL141051; Ascaris lumbricoides retrotransposon—translation of L22247; Caenorhabditis elegans retrotransposon—translation of Z38112; Gypsy—GNFFG1; Ted—M32662; Mdg1—X59545; Osvaldo—AJ133521; Ulysses—X56645; Maggy—L35053; Sushi—AF030881; Ty3—M34549; Blastopia—Z27119; Mdg3—X95908; Cer1—U15406; human foamy virus—Y07725; feline leukemia virus—AAA93092

 
Distribution and Copy Number of Jule-Related Sequences in Teleosts
The distribution and the copy number of Jule were investigated by Southern blot hybridization analysis in species covering about 180 Myr of teleost evolution and in a nonteleost fish, the sturgeon Acipenser sturio (Benton 1990Citation ; Nelson 1994Citation ; fig. 5 ). The 1.8-kb insert of plasmid pROST56-214 (see Materials and Methods and fig. 1 ) was used as a Jule-specific probe. Hybridization patterns obtained for X. maculatus Rio Jamapa suggested that female diploid genomes (XX) have two 7.5-kb fragments (intron 1 of the X allele of the Xmrk proto-oncogene), two 4.8-kb fragments (3' of the X allele of the Xmrk oncogene), and two other pairs of fragments (according to their relative intensity to the 4.8- and 7.5-kb fragment pairs). Male diploid genomes (XY) have only one 4.8-kb fragment (X chromosome), with the second 4.8-kb fragment being replaced by a 7.3-kb fragment (3' of the Y allele of the Xmrk oncogene), and they have only one 7.5-kb fragment (X chromosome) and the two additional pairs of fragments. Hence, hybridization patterns observed for X. maculatus Rio Jamapa males and females are consistent with the presence of seven copies per diploid genome in males and eight copies per diploid genome in females. Additional males and females were tested: they all presented hybridization patterns identical to those shown in figure 5 (not shown).



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Fig. 5.—Southern blot hybridization analysis of the distribution and the copy number of Jule in teleosts. All genomic DNAs were cut with EcoRI. Filters were washed with 2 x SSC–1% SDS at 50°C (low stringency). The Oryzias latipes filter was overexposed compared with the other filters. f = females; m = males; RJ = population Rio Jamapa; RU = population Rio Usumacinta

 
Jule-related elements were detected by Southern blot hybridization in another population of X. maculatus (population Rio Usumacinta) and in other Xiphophorus species (X. milleri and X. helleri) (fig. 5 ). Some differences in fragment size and intensity between males and females were detected in Xiphophorus species other than X. maculatus. These differences, observed in highly inbred laboratory stocks, might be sex-specific, but could also result from sex-independent insertion polymorphism. More individuals have to be analyzed to clarify this point. Signals were also obtained for other members of the family Poeciliidae, including P. mexicana, G. affinis, G. falcatus, and P. gracilis (fig. 5 ) and Phallichthys amates, Heterandria bimaculata, Heterandria formosa, Poecilia latipinna, Poecilia formosa, and Girardinus metallicus (not shown). Jule-related elements were also revealed in Fundulus sp. and O. latipes (medakafish), which belong to different families but are both within the order Cyprinodontiformes, which includes the Poeciliidae (Nelson 1994Citation ). Signals were observed in O. niloticus (order Perciformes) as well. In all fish species in which signals were detected, the copy number of Jule-related sequences was small (less than 10 per haploid genome; fig. 5 ).

No signal was detected in more divergent fishes, including O. mykiss (rainbow trout), E. lucius (pike), D. rerio (zebrafish), C. carpio (carp), A. anguilla (European eel), and A. sturio (sturgeon) (fig. 5 ). This indicates either that Jule is not present in theses fishes or that the level of sequence similarity is too low to be detected by Southern blot hybridization.

Using Jule amino acid sequence as a query, database searches identified related elements in the genomes of other teleost fishes, including the zebrafish D. rerio (fig. 6A ) and the two genome project pufferfishes, F. rubripes (Japanese pufferfish; fig. 6B ) and T. nigroviridis (freshwater pufferfish; fig. 6C-1 and C-2 ). The sequence from D. rerio is the conceptual translation product of an embryo-expressed sequence tag (accession number AA495349) showing 56% amino acid identity and 71% amino acid similarity to a part of Jule reverse transcriptase (fig. 6A ). The relatively low level of nucleotide identity with Jule (58%) explains why the D. rerio element was not detected by Southern blot hybridization.



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Fig. 6.—Identification of Jule-related sequences in public databases. Identical residues are shown in black, and conservative substitutions are shown in gray (drawn using MacBoxshade). A, Danio rerio, conceptual translation of EST AA495349. B, Fugu rubripes, consensus translation of sequences aD1 and aF1 from cosmid 161D05 (http://fugu.hgmp.mrc.ac.uk). C-1, Tetraodon nigroviridis, consensus translation from the Tetraodon genome project (www.genoscope.cns.fr), including sequences AL252617, AL347043, and AL211244. C-1, Tetraodon nigroviridis, consensus translation from the Tetraodon genome project (www.genoscope.cns.fr), including sequences AL186592, AL176544, and AL226469. Jule: consensus translation of AF278691 and AF278692

 
Sequences similar to Jule were also detected in the Fugu rubripes genome project (Elgar et al. 1996Citation ). The amino acid sequence shown in figure 6B (putative translation product of sequences aD1 and aF1 from cosmid 161D05; http://fugu.hgmp.mrc.ac.uk) displays 55% identity and 67% similarity to a region of Jule from the C-terminal domain of the reverse transcriptase to approximately 30 aa downstream of RNase H motif DAS. A Jule-related element was also detected in T. nigroviridis. By assembling sequences generated by the Genoscope sequencing center (www.genoscope.cns.fr), an almost complete Jule-like element from T. nigroviridis could be reconstructed. Unfortunately, Jule-related sequences could not be assembled into one unique contig, but could be assembled into a 5' contig (including sequences AL252617, AL347043, and AL211244 and containing the 5' LTR and the 5' part of the gag region; putative translation product in fig. 6C-1 ) and a 3' contig (including sequences AL186592, AL176544, and AL226469, from the 3' part of gag to the 3' LTR; putative translation product in fig. 6C-2 ). The 5' and 3' contig conceptual translation products display 45%/53% and 44%/54% amino acid identities/similarities, respectively, to the corresponding regions of Jule. The T. nigroviridis element is delimited by two 148-nt LTRs, and its putative Gag protein contains two CX2CX4HX4C zinc finger domains (fig. 6C-2 at the beginning of the Tetraodon sequence). Phylogenetic analysis indicated that the D. rerio and F. rubripes sequences are more related to Jule, but could not determine if the T. nigroviridis sequence is more related to Jule or to A. gambiae retrotransposon 1 (not shown). No Jule-related element could be detected by database searching in other vertebrate lineages.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Jule from the platyfish X. maculatus is, to our knowledge, after the Sushi element of the pufferfish F. rubripes (Poulter and Butler 1998Citation ), the second complete LTR retrotransposon from the Ty3/Gypsy superfamily described in the vertebrate lineage. Jule is clearly much more related to some insect and nematode retrotransposons than to Sushi. Both fish retrotransposons belong to different families of Ty3/Gypsy retrotransposons: while Sushi is member of the Ty3 family, Jule is included within the Mag family. Jule is the first member of the Mag family to be discovered in vertebrates.

In addition to known members of the Mag family, including Mag from the silkworm B. mori (Michaille et al. 1990Citation ; Garel, Nony, and Prudhomme 1994Citation ) and SURL from the sea urchin T. gratilla (Springer, Davidson, and Britten 1991Citation ), we identified additional elements in the nematodes C. elegans (this observation has been already made by others, e.g., Malik and Eickbush [1999Citation ]) and A. lumbricoides and in the malaria vector, the African mosquito A. gambiae.

In classical phylogenies, the sea urchin T. gratilla is more related to the fish Xiphophorus (both are deuterostomes) than to nematodes and insects, A. gambiae is more related to B. mori (both insects) than to Xiphophorus, and C. elegans is more related to A. lumbricoides (both nematodes) than to A. gambiae (http://www.ncbi.nlm.nih.gov/Taxonomy). Hence, there are some discrepancies between the phylogeny of retrotransposons of the Mag family (fig. 4 ) and the phylogeny of their host genomes. This phenomenon, which has been already observed for some other transposons (Capy, Anxolabéhère, and Langin 1994Citation ; Cummings 1994Citation ), might result from differences in evolutionary rates. Sequences with a higher rate of evolution can typically be "pulled down" to the root in a tree and will give the impression that they diverged earlier from a lineage than they evolutionarily did. Nevertheless, comparison of protein distances between Mag-related retrotransposons and other elements revealed that the members of the Mag family included in this study have evolved roughly at the same rate (not shown). More likely, discrepancies between Mag-related retrotransposon and host phylogenies might be explained by the presence of ancient retrotransposon lineages, which diverged before their host genomes did (Capy, Anxolabéhère, and Langin 1994Citation ; Cummings 1994Citation ). Some members of these lineages might have been lost or not detected. This hypothesis is consistent with the presence of two phylogenetically distinct A. gambiae elements, each of them more related to elements from other organisms than to the other A. gambiae sequence (fig. 4 ). This strongly suggests that the Mag family of Ty3/Gypsy retrotransposons is polyphyletic. Finally, horizontal interspecific gene transfer of Mag-related retrotransposons could potentially have influenced the topology of the phylogeny of the Mag family. SURL elements can be transmitted horizontally (Gonzalez and Lessios 1999Citation ).

As reported for Mag and SURL elements, no envelope gene or domain could be detected in the other Mag-related elements identified in this work, and neither GPY/F motif nor chromodomain (Malik and Eickbush 1999Citation ) could be found in their integrase domain. The sizes of the LTRs in the Mag family vary between 77 nt for Mag (Michaille et al. 1990Citation ; Garel, Nony, and Prudhomme 1994Citation ) and 254 nt for SURL (Springer, Davidson, and Britten 1991Citation ). The size of the intervening region between both LTRs is relatively constant (between 4,340 nt for Jule and 4,750 nt for SURL). In the majority of Mag-related retrotransposons, including Jule and the related element from T. nigroviridis, the A. lumbricoides element, Mag, and SURL, the Gag protein contains two successive zinc finger–like motifs. One exception is the C. elegans element, in which only one zinc finger was detected. This was not a specificity of the copy chosen for analysis, because related elements from C. elegans showed only one motif as well. According to the phylogeny of Mag-related elements (fig. 4 ), the most simple explanation is that the last ancestor of the Mag family had two zinc finger–like motifs and that one of them has been lost in C. elegans. As observed for Mag (Garel, Nony, and Prudhomme 1994Citation ) and for other retrotransposons, integration of Jule generates a 5-nt duplication of the target sequence. The low similarity between the target sequences of both Jule elements (CTCTA and GTCTT) suggests low or no sequence specificity, as reported for Mag (Garel, Nony, and Prudhomme 1994Citation ).

Jule-like sequences were detected in all members of the family Poeciliidae tested so far and in other Cyprinodontiformes (Fundulus sp. and O. latipes). Related elements were also found in less related fishes, including O. niloticus, the two pufferfishes F. rubripes and T. nigroviridis, and the zebrafish D. rerio. Hence, Jule-related sequences are present in teleost species that diverged about 100 MYA (Benton 1990Citation ). No related sequences could be identified by database searching in other vertebrate lineages. Considering the large amount of sequences available, particularly for Homo sapiens (more than 80% estimated coverage of the human genome with working draft sequences at the time of analysis), this suggests that the different retrotransposon lineages constituting the Mag family are absent or present at very low copy numbers in certain vertebrate lineages. Alternatively, they might be present in special genomic niches that have been only poorly sequenced to date because of their high content of repetitive DNA. Although there is no evidence for such a phenomenon, Jule-related retrotransposons might also have been reintroduced through horizontal transfer from a nonvertebrate species into the teleost lineages at least 100 MYA.

While Jule-2 is more corrupted, the low level of corruption of Jule-1 suggests that Jule-1 is the result of a recent retrotransposition event. Although Jule-1 and Jule-2 are both associated with a version of the Xmrk gene, their different locations (in intron 1 of the Xmrk proto-oncogene and 3' of the Xmrk oncogene, respectively) and their different target site duplication sequences indicate that they are the result of two different retrotransposition events. The high degree of nucleotide identity between Jule-1 and Jule-2 suggests again recent activity of a Jule element. Generally, the only selective pressure conserving retrotransposon enzymatic functions during evolution is their own ability to transpose. The low copy number of Jule retrotransposons detected by Southern blot hybridization in Poeciliidae and some other teleost species suggests that this retrotransposon did not maintain its functionality through frequent retrotransposition in its host genomes. If Jule did it, there is a counterbalancing mechanism of elimination maintaining a low copy number of the retrotransposon. An alternative hypothesis is the horizontal transfer of Jule between fishes. The SURL elements, which, like Jule, belong to the Mag family of retrotransposons, can be transmitted horizontally between echinoid species (Gonzalez and Lessios 1999Citation ).

There are only three to four copies of Jule per haploid genome in X. maculatus (depending on whether the haploid genome considered contains the Y or the X sex chromosome). Curiously, two of these copies, which resulted from two different retrotransposition events, are physically associated with the Xmrk genes: one copy is associated with the Xmrk oncogene, and a second one is associated with one allele of the Xmrk proto-oncogene. Both are in the same subtelomeric region of the sex chromosomes of the platyfish. Both Jule-1 (X-specific) and Jule-2 (presenting sex-specific differences according to Southern blot analysis) insertions are closely linked to the sex determination locus: analysis of about 200 individuals of one strain of X. maculatus showed absence of recombination between both Jule insertions and the sex determination locus (unpublished data). We have strong evidence of the accumulation of other repetitive elements in this region (Volff et al. 1999Citation ; Volff, Körting, and Schartl 2000Citation ; unpublished data). This region is also prone to other DNA rearrangements, including deletions, insertions, and DNA amplification, with some of them being specific for either the X or the Y sex chromosome (Schartl et al. 1999Citation ; Nanda et al. 2000Citation ; unpublished data). Such sex-linked rearrangements might contribute to the initiation of divergence between the primitive sex chromosomes of X. maculatus and to recombinational isolation of the sex-determining locus. Such a phenomenon is reminiscent of the accumulation of certain retrotransposons on the Y chromosome of some Drosophila species (Steinemann and Steinemann 1997Citation ; Chalvet et al. 1998Citation ).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank G. Schneider, H. Schwind, and P. Weber for fish maintenance; S. Chen and Y. Hong (Würzburg, Germany), S. Kirilchik, and M. Grachev (Irkutsk, Russia) for fish organs and DNA; K. T. Cuong and A. K. Meyer for sequencing, J. Altenbuchner (Stuttgart, Germany) for the transposon mutagenesis system; and the Fugu, Tetraodon, and Anopheles genome projects for making sequences available to the public. This work was supported by grants 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
 
Pierre Capy, Reviewing Editor

1 Abbreviations: aa, amino acids; LTR, long terminal repeat; nt, nucleotides. Back

2 Keywords: SURL Gag Pol Xmrk sex chromosomes 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


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Accepted for publication September 24, 2000.