Download The Non-LTR Retrotransposon Rex3 from the Fish Xiphophorus is

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
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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
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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).
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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
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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