Department of Microbiology, College of Medicine, Catholic University of Korea, Seoul, Korea
Department of Molecular Parasitology, Sungkyunkwan University School of Medicine, Suwon, Korea
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Abstract |
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Introduction |
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With the cumulative data on retrotransposons, various studies have discussed the probable evolutionary course of retrotransposons, including studies of long-terminal-repeat (LTR) family, non-LTR family, and exogenous retroviruses (Xiong and Eickbush 1990
; Malik, Burke, and Eickbush 1999
; Malik and Eickbush 1999
). However, the phylogenetic relationship of diverse retrotransposons remains ambiguous, mainly with regard to factors concerning the branching patterns in phylogenetic trees, such as the formation of polytomy (Malik and Eickbush 1999
) and polyphyletic distribution (Malik and Eickbush 1999
; Marín and Lloréns 2000
). Thus, the number of ancient classes responsible for diverse clades of retrotransposons and the extent of possible horizontal transfer between different species are difficult to define. Such ambiguities are likely to arise from limitations in sequence information on retrotransposons and unbalanced sampling from each taxon.
There have been a number of reports on the LTR retrotransposons of animals such as nematodes (Felder et al. 1994
; Bowen and McDonald 1999
), insects (Lindsley and Zimm 1992
; Biessmann et al. 1999
; Abe et al. 2000
), echinoderms (Britten et al. 1995
), and fish (Poulter and Butler 1998
). In Platyhelminthes, however, Gulliver of Schistosoma japonicum (Laha et al. 2001
) is the only full-length LTR retrotransposon so far described in the phylum, although Arkhipova and Meselson (2000)
have recently reported a segmental sequence of an LTR retrotransposon in Dugesia. Moreover, the sequence of Gulliver is corrupted, even though its expression has been demonstrated at the level of transcription by reverse transcription-PCR (RT-PCR) (Laha et al. 2001
). Thus, currently there are no reports on uncorrupted LTR retrotransposons, which may play a significant role in the formation of genomes in Platyhelminthes, including trematodes.
Retrotransposons introduce variations through their heterogeneous integration and subsequent sequence divergence, and these polymorphic regions can be identified as randomly amplified polymorphic DNAs (RAPDs) by arbitrarily primed PCR (AP-PCR) (Abe et al. 1998
). In the present study, we attempted to identify retrotransposons from the genome of Clonorchis sinensis, an important human liver fluke in East Asia, based on the analysis of RAPD sequences. By screening variable genetic regions from individual C. sinensis using the AP-PCR method, a retrotransposon, named Clonorchis sinensis Retrotransposon 1 (CsRn1), was isolated as the second complete but the first uncorrupted LTR retrotransposon identified in the phylum Platyhelminthes. Structural and genomic analyses of CsRn1 and its phylogenetic relationship with other Ty3/gypsy-like LTR retrotransposons are presented.
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Materials and Methods |
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AP-PCR and Cloning of Individual Worm-Specific Products
Genomic DNAs extracted from individual worms were used for the amplification of worm-specific RAPD regions by PCR under low-stringency conditions. The following arbitrarily designed primers were used in the PCR reactions: AP-1 (5'-GATCCGTTCA-3'), AP-3 (5'-ACCCATACCC-3'), B4 (5'-GGACTGGAGT-3'), B5 (5'-TGCGCCCTTC-3'), B8 (5'-GTCCACACGG-3'), B12 (5'-CCTTGACGCA-3'), B13 (5'-TTCCCCCGCT-3'), F6 (5'-GGGAATTCGG-3'), and F15 (5'-CCAGTACTCC-3'). The reaction mixtures included 40 ng of genomic DNA, 1.25 µM of primers, 0.2 mM each of dATP, dGTP, dCTP, and dTTP, and 1.25 U of Taq polymerase (Takara, Shiga, Japan) in a total reaction volume of 20 µl. PCR conditions were as follows: 32 cycles of 1 min at 94°C, 1 min at 37°C, and 2 min at 72°C, and a final extension of 10 min at 72°C. The reproducibility of the results was tested by repeating the reactions under identical conditions three times. The PCR products were fractionated by electrophoresis on agarose gels and stained with ethidium bromide. Individual-specific bands were recovered from agarose gels, reamplified with the corresponding primers, and then cloned into pGEM-T Easy Vector (Promega) for nucleotide sequencing.
Southern Blot Hybridization
Five micrograms of genomic DNAs isolated from C. sinensis adult worms were digested with restriction enzymes. After being fractionated through 0.8% agarose gels, the DNAs were blotted onto nylon membrane (Hybond-N+; Amersham Pharmacia Biotech, Uppsala, Sweden) by capillary action in 10 x standard saline citrate (SSC). The blots were hybridized with probes enzymatically labeled with the ECL Direct Labeling Kit (Amersham Pharmacia Biotech). The labeling and hybridizing conditions were according to the manufacturer's instructions. The membranes were washed twice in 6 M urea, 0.4% sodium dodecyl sulfate, and 0.1 x SSC at 42°C for 20 min and twice in 2 x SSC at room temperature for 5 min.
Dot-Blot Analysis
Ten micrograms of genomic DNA were blotted onto nylon membrane according to the standard procedure of dot-blot analysis (Sambrook, Fritsch, and Maniatis 1989
). Two identical membranes were prepared, of which one was hybridized with probe for repetitive sequences and the other was hybridized with that for cysteine protease as a single copy control (GenBank accession number AF271091). The probe for protease was amplified from C. sinensis genomic DNA with the CsCP3-S1 (5'-GCTGGACTCCGACTACCCATATG-3') and CsCP3-R3 (5'-GGTTTAAACGATTGTGCATCGC-3') primers. Probe labeling and hybridizing conditions were same as those for Southern blot hybridization. The intensities of signals were measured using the LAS-1000plus system (FUJIFILM, Tokyo, Japan).
Construction and Screening of Genomic DNA Library
DNAs from adult worms were partially digested with Sau3AI (Takara). DNA fragments of 923 kb were recovered by ultracentrifugation onto sucrose gradients, purified, and then cloned into lambda FIX II vector predigested with XhoI (Stratagene, La Jolla, Calif.). The constructs were packaged into lambda particles using Gigapack III Gold-11 packaging extract. The unamplified libraries were screened with DNA probe labeled with the ECL labeling kit. The conditions for plaque-lift hybridization were identical to those for Southern blot hybridization. The inserts of lambda clones were amplified by long PCR (Chen, Fockler, and Higuchi 1994
) using primers designed from vector regions (5'-CTAATACGACTCACTATAGGGCGTCG-3' and 5'-CCCTCACTAAAGGGAGTCGACTCG-3') and LA Taq polymerase according to the standard cycle conditions (Takara). The amplified products were digested with restriction enzymes and were then cloned into pBluescript II SK(-) phagemid (Stratagene) for nucleotide sequencing.
Screening of cDNA Library by PCR Methods
A cDNA library of adult C. sinensis was constructed in ZAP II vector using a cDNA synthesis kit (Stratagene) according to the manufacturer's instructions. The library was amplified and was then used in standard PCR reactions for the detection of mRNA transcripts. The PCR products were cloned into pGEM-T Easy Vector for sequencing.
Sequence Analysis
The nucleotide sequences were automatically determined with an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, Calif.) and a BigDye Terminator Cycle Sequencing Reaction Kit (Perkin Elmer Corporation, Foster City, Calif.). To ensure the accuracy of sequencing reactions, sequences of single strands from five clones of vector-ligated DNA fragments were determined. For PCR products obtained during cDNA library screening, nucleotide sequences from both strands were determined. After sequencing, homology searches were performed against the nonredundant database at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nhm.gov/) using BLASTN and BLASTX (Altschul et al. 1997
). The REPEAT program in the GCG package, version 8. (University of Wisconsin), was used to determine the direct repeat sequences. The putative open reading frames (ORFs) were predicted by GeneScan (Burge and Karlin 1997
), GeneMark (Borodovsky and Lukashin, unpublished; http://genemark.biology.gatech.edu/GeneMark/), and ORF Finder (NCBI). A search for the functional protein domains was performed using ProDom (Gouzy, Corpet, and Kahn 1999
) and ProfileScan (Gribskov, McLachlan, and Eisenberg 1987
).
Phylogenetic Analysis
The nucleotide sequences were aligned with ClustalX (Thompson et al. 1997
). After optimizing the sequence alignments using the PHYDIT program (Chun 1995
), divergence values were calculated and a dendrogram was drawn using the programs DNADIST and NEIGHBOR, respectively, of PHYLIP (Felsenstein 1993
). Based on the previous reports (Xiong and Eickbush 1990
; Malik and Eickbush 1999
), reverse transcriptase (RT), RNase H (RH), and integrase (IN) domains in pol proteins were defined from alignments using CLUSTAL W (Thompson, Higgins, and Gibson 1994
), and amino acid sequences of the three domains were combined for the phylogenetic analysis of LTR retrotransposons. After aligning the combined sequences with ClustalX and optimizing the alignment with GeneDoc (Nicholas and Nicholas 1997
), a phylogenetic analysis was performed using PROTDIST and NEIGHBOR of PHYLIP. The trees were displayed by TreeView (Page 1996
), and the statistical significance of branching points was evaluated with 1,000 random samplings of the input sequence alignments using SEQBOOT.
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Results |
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The Mobile Activity of the CsRn1 Master Copy
The genomic distribution of the element was found to be heterogeneous among individuals of C. sinensis, which supports the recent expansion of CsRn1 (fig. 7A
). Based on this finding, we attempted to examine the presence of CsRn1 transcripts in the total RNA molecules extracted from the adult worms by Northern blot analysis but failed to detect any signals when the blots were probed with P-B13 probe (data not shown). We then performed PCRs with higher sensitivity using a cDNA library as template and four primer sets covering the full-length of mRNA transcripts (see fig. 7B
and its legend). As shown in figure 7B
, 3'-end and P-B13 regions were well amplified, whereas 5'-end and RT regions were not amplified or weakly amplified, possibly due to the incomplete extension of cDNAs from 3' ends during the construction of the cDNA library.
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The Evolutionary Relationship of CsRn1 with Other Retrotransposons
The amino acid sequence of RT encoded in CsRn1 showed strong homology with that of RT in many Ty3/gypsy-like LTR retrotransposons, particularly in Kabuki (identity value of 63%) and AE003787 (58%), and a similar pattern of homology was also found with the sequences of IN (fig. 4
). Moreover, the nucleotide sequence of CsRn1 exhibited homology with the DNA sequences of the two elements over a relatively long range of nucleotide positions (Kabuki, 53.6% identity in 2,972 nt; AE003787, 53.5% in 3,583 nt). In view of these observations, we performed a phylogenetic analysis using amino acid sequences of pol proteins in order to gain further understanding of the relationship of CsRn1 with other Ty3/gypsy-like LTR retrotransposons. In addition to RT, the amino acid sequences of RH and IN were selected for this analysis to increase the resolution (Malik and Eickbush 1999
).
In a previous report, Malik and Eickbush (1999)
divided Ty3/gypsy-like LTR retrotransposons into eight distinct clades. As shown in figure 8
, the members of the eight clades were well separated in a tree constructed by the UPGMA method. Interestingly, however, CsRn1 formed a previously undetected, tightly conserved clade with Kabuki and AE003787. A similar clustering pattern was observed in a tree constructed with the neighbor-joining algorithm (data not shown), and the statistical significance of the branching points was well supported by bootstrap analysis. As members of a new clade (designated the CsRn1 clade), the three elements shared a number of common features, such as a CHCC Gag motif, a TSD of 4 bases (in the case of Kabuki, TSD cannot be determined; see Abe et al. 2000
), and sequence conservations in the PBS and PPT (fig. 3
), as well as functional protein domains (fig. 4 ).
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Discussion |
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For expansion, retrotransposons are transcribed by host RNA polymerase and then reverse-transcribed by their own reverse transcriptase. Because these two enzymes have no proofreading capacity (Varmus and Brown 1989
), cDNAs produced during the process of transposition tend to acquire random base substitutions. These base substitutions frequently inactivate the progeny copies, and the resulting "dead-on-arrival" (DOA) copies with no mobile activities are likely to be subjected to neutral evolution, through which the accumulation of sequence variations within the DOA copies is accelerated (Petrov, Lozovskaya, and Hartl 1996
). Thus, together with the low fidelity of RT, the neutral evolution of the inactive DOA copies may have an additional effect on sequence divergence among individual genomes of a species. In this study, a variable genomic region among individuals was successfully identified by AP-PCR-based RAPD analysis as CsRn1 LTR retrotransposon-related sequence.
During an evolutionary time, a particular subset of retrotransposons expands differentially, rather than simultaneously, from other variant subsets with selective advantage for expansion (Clough et al. 1996
). Thus, a recently expanded master copy can be distinguished as the subset with the largest population (Boissinot, Chevret, and Furano 2000
) and with low levels of sequence divergence among its members (Medstrand and Mager 1998
). In addition to these characteristics, high sequence identity between flanking LTRs of individual copies can be accepted as a hallmark of their recent integration in cases of LTR retrotransposons, since a pair of LTRs use the same sequences as templates for their replications (Dangel et al. 1995
). CsRn1 copies of group G satisfied all of these criteria (table 1
and fig. 6
), suggesting the role of group G as an active master copy, and the preserved mobile activity was confirmed by the uncorrupted ORF, heterogeneous distribution among individual genomes (fig. 7A
), and the presence of mRNA transcripts of CsRn1 (fig. 7B
).
The coding capacity of CsRn1 suggests that the element is a member of metaviruses, which have no env gene (Pringle 1999
). Although no significant homology to other LTR retrotransposons at the nucleotide sequence level was found throughout the whole unit, sequence motifs for the synthesis of double-stranded cDNA (PBS and PPT) showed strong identity with those of Kabuki (B. mori) and AE003787 (D. melanogaster) (fig. 3
). Moreover, the amino acid sequences in the unusual CHCC Gag motif, RT, and IN were well conserved in the three elements (fig. 4
). With these shared properties, a phylogenetic analysis suggested that CsRn1 formed an ancient, previously undetected clade of Ty3/gypsy-like LTR retrotransposons found in insects and trematodes (CsRn1 clade; fig. 8
). The nucleotide sequences of Kabuki and AE003787 in their putative ORF regions are corrupted, which inactivates the elements and gives rise to low copy numbers (the number of Kabuki was estimated as eight in the haploid genome; Abe et al. 2000
). However, several copies of CsRn1 were uncorrupted and showed maintained mobile capacity (fig. 7
), suggesting that the element might have acquired high copy numbers in C. sinensis genomes (more than 100 per haploid genome; fig. 5
) through its continuous expansion.
Although they had a phylogenetically close relationship, differences in the numbers of ORFs and no significant homology in nucleotide sequences were observed among the members of the new clade, which reduces the probability of horizontal transfer between insects and trematodes in the recent past. Thus, it is likely that these elements evolved from a common ancestor that was present in the common progenitor of insects and trematodes or was transferred from insects to trematodes, or vice versa, during the early stage of the divergence of the two taxa. However, the possibility of horizontal transfer between insects and trematodes is uncertain because few cases, only within similar species, have been reported (Gonzalez and Lessios 1999
; Jordan, Matyunina, and McDonald 1999
). The isolation of further elements belonging to the CsRn1 clade in insects and trematodes or the finding of an errantivirus(s) that is phylogenetically related to the clade (Malik and Eickbush 1999
) will be helpful in understanding the detailed evolutionary course among the members of the clade.
Only a few data on LTR retrotransposons are available for the Platyhelminthes, despite the large content of repetitive elements in their genomes (see Regev, Lamb, and Jablonka 1998
and references therein). Model organisms such as D. melanogaster, A. thaliana, and S. cerevisiae, commonly used in previous studies, have genome structures with relatively low complexity and repetitive elements with low copy numbers, which makes it difficult to estimate the actual significance of retrotransposons in the complex genome of the animal. Thus, the present study using a trematode provides advantages for the study of retrotransposons with small but complex genomes. We confirmed the presence of partial sequences similar to those of CsRn1 in other trematodes, S. mansoni (fig. 7C
) and Paragonimus westermani (unpublished data). These results reflect the presence of a unique LTR retrotransposon family belonging to the CsRn1 clade in the lower animal taxa which may play significant roles in the evolution of genomes. Our results concerning an active LTR retrotransposon and its related elements will broaden the current knowledge on LTR retrotransposons and provide a clue for further studies on the evolutionary origin of diverse reverse-transcribing elements.
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Supplementary Material |
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A sequence alignment of multiple CsRn1 copies was deposited in GenBank in linked form to each nucleotide set, of which accession numbers are presented above, and the alignment of pol proteins used for phylogenetic analysis in this work was deposited in linked form to the nucleotide sequence of CsRn1-4 (AY013569).
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Acknowledgements |
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Footnotes |
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1 Abbreviations: IN, integrase; PBS, primer-binding site; PPT, polypurine tract; PR, protease; RT, reverse transcriptase; TSD, target site duplication; RH, RNase H.
2 Keywords: Clonorchis sinensis
trematode
LTR retrotransposon
AP-PCR
RAPD
master copy
3 Address for correspondence and reprints: Mun-Gan Rhyu, Department of Microbiology, College of Medicine, Catholic University of Korea, Seoul 137-701, Korea. rhyumung{at}cmc.cuk.ac.kr
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