Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo, Japan
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Abstract |
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Introduction |
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The telomeres of the silkworm, Bombyx mori, consist of telomeric repeats (TTAGG)n and harbor many types of non-LTR retrotransposons (Okazaki, Ishikawa, and Fujiwara 1995
; Takahashi, Okazaki, and Fujiwara 1997
; Fujiwara et al. 2000
). More than 2,000 copies of these retrotransposons are inserted into the repeats in a highly sequence-specific manner. They are classified into two groups, TRAS and SART, based on their insertion sites and directions. We have speculated that more than eight families of TRAS or SART elements are present in the silkworm telomere, while the fine structures of only two families, TRAS1 and SART1, have been studied completely.
Most nonlong terminal repeat (non-LTR) retrotransposons are randomly integrated into the host genome, while some have preferable target sites. Several non-LTR elements in Drosophila, Jockey (Priimagi, Mizrokhi, and Ilyin 1988
), F (Minchiotti and Di Nocera 1991
), and I (McLean, Bucheton, and Finnegan 1993
), seem to have no target specificity. The human L1 element L1Hs also has preferable target sequences that are not so strictly defined (Jurka 1997
; Cost and Boeke 1998
). Some non-LTR retrotransposons, however, have a very restricted target in the genome. R1 and R2 are inserted at specific sites in 28S rDNA of insects (Jakubczak, Burke, and Eickbush 1991
). Tx1L of Xenopus laevis locates in a specific site within another family of transposable elements (Tx1D) (Christensen, Pont-Kingdon, and Carroll 2000
). RT1 and RT2 are inserted into the specific sites of 28S rDNA of both A. gambiae and Anopheles arabiensis (Paskewitz and Collins 1989
; Besansky et al. 1992
).
Recent studies have revealed that endonuclease domains encoded in non-LTR retrotransposons are involved in target recognition and cleavage (Feng et al. 1996
). Endonucleases in the N-terminal region of ORF2 (L1Hs [Cost and Boeke 1998
], R1Bm [Feng, Schumann, and Boeke 1998
], and Tx1L [Christensen, Pont-Kingdon, and Carroll 2000
]) and in the C-terminal region of R2 ORF (Yang, Malik, and Eickbush 1999
) have been shown to cut their target sequence at specific sites. Recently, we also found that the TRAS1 endonuclease of the silkworm could cut the (TTAGG)n at specific sites (Anzai, Takahashi, and Fujiwara 2001
). These observations indicate that the endonuclease domain makes a first cleavage on the bottom strands of the target sequences. However, it is still unknown what sequences in the EN (endonuclease) domain or any other regions within ORFs of retrotransposons are involved in target site selection or recognition.
Amino acid sequence comparison may reveal the conserved elements only among retrotransposons that have the same target sequences, leading one to speculate on the putative sequences involved in target specificity. In this study, we screened different classes of TRAS elements from the silkworm and from two other Lepidoptera. Through a comparison of amino acid sequences from EN to RT (reverse transcriptase) domains in ORF2 among the non-LTR retroelements, we found highly conserved regions only among TRAS-like elements. One of these resembles the Myb-like DNA binding structure.
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Materials and Methods |
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Cloning, PCR Amplification, and Sequence Analyses
Novel TRAS families of B. mori have been screened from a genomic lambda phage library which was used for TRAS1 isolation (Okazaki, Ishikawa, and Fujiwara 1995
). Phage clones containing (TTAGG)n repeats were isolated with a 32P-labeled (TTAGG)5 probe. In addition to TRAS1, we found five classes of TRAS families, named TRAS3, TRAS4, TRASY, TRASW, and TRASZ, among (TTAGG)n-bearing phage clones. The complete sequence of TRAS3 was determined. In TRAS4, the region from the EN to the RT domain was amplified by PCR (see below) and sequenced. Only junction regions between retrotransposons and (TTAGG)n were analyzed for TRASY, TRASZ, and TRASW. Since several regions in EN and RT domains are highly conserved among non-LTR retrotransposons, we amplified the region from EN to RT (approximately 70% of the region of RT) by PCR from respective TRAS families in the silkworm and other lepidopteran insects. We designed some primers based on the consensus-degenerate hybrid oligo-nucleotide primer (CODEHOP) strategy (Rose et al. 1998
). Primers used for PCR were as follows: TR4QSAG, 5'-AGGGCGCAAGATCTTCCAAAGCGCTGGCCC-3'; TR5GYKG, 5'-GTTCTTCAACAGTGAGGGGATATAAAGGAGC-3'; TR6SLEN, 5'-GCCTGGCAATACTCTCCACTGTTTTCGAGAC-3'; GTVK, 5'-GGGACTGTNAAAGCNGCNAT; CH-VVGI, 5'-ACCACCAACAACATCGTCGTRGTNGRRRTC-3'; CH-FADD, 5'-GTCTCCGTCGAAAACCAGGACCACRTCRTCNGCRAA-3'. To amplify respective classes of TRAS elements, the following primer sets were used in PCR reaction: TRAS4, TR4QSAG + CH-FADD (annealing at 61°C); TRAS5, TR5GYKG + CH-FADD (51°C); TRAS6, GTVK + TR6SLEN (51°C); TRAS in D. japonica, CH-VVGI + CH-FADD (51°C); TRAS in S. cynthia, GTVK + CH-FADD (51°C). PCR was performed for 35 cycles of 94°C for 45 s, 5161°C for 45 s, and 72°C for 90 s. Amplified PCR products were cloned into the pGEM(R)-T Easy vector (Promega) and sequenced with ABI-310 DNA sequencer (PE Applied Biosystems).
Structural Prediction
The domains from EN to RT of non-LTR retrotransposons were aligned using the multiple alignment options in CLUSTAL W (Thompson, Higgins, and Gibson 1994
). The phylogenetic tree of retrotransposons was based on the sequence of EN to RT domains using the neighbor-joining method (Saitou and Nei 1987
). Secondary-structural prediction was carried out with DNASIS, version 3.7 (Hitachi).
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Results and Discussion |
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From more than 100 positive clones, 8 were selected and subcloned into plasmid vectors for further characterization and sequence analysis (fig. 1 ). Based on the structural difference in the junction regions between the 5' end of retroelements and the target (TTAGG/CCTAA)n, we identified six new families, which we classified into two large groups. Five families, named TRAS3, TRAS4, TRASY, TRASW, and TRASZ, are oriented distal to the telomeric end and are adjacent to CC of the (CCTAA)n telomeric strand, similar to TRAS1 (fig. 3A ). In contrast, one family, named SART2, is oriented in the reverse direction of the TRAS groups and is inserted between the T and A nucleotides of (TTAGG)n, similar to SART1 (fig. 1 ). Restriction mapping, partial sequencing, and hybridization studies on these lambda phage clones revealed that different classes of TRAS or SART elements were clustered between short stretches of the telomeric repeats. Based on the numbers of positive clones in plaque hybridization with each class of retroelement as a probe, we speculate that more than 2,000 copies of TRAS and SART elements, which average 7.5 kb, occupy 3% of the silkworm genome: SART1, 600; TRAS1, 300; TRAS3, 300; other TRAS and SART, 50200 per haploid genome (data not shown). As can be easily imagined from the structure of the phage clones in figure 1 , the subtelomere region of the silkworm may consist of alternate tandem arrays of retroelements (more than 40 copies at each end) and short (TTAGG/CCTAA)n sequences.
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We also compared the 3'-end structures of each class of TRAS (fig. 3B
). While the data are inconclusive, nucleic acid similarity between two TRAS3 clones, TRAS3-1 (complete unit) and p3-3L, in the 3'-terminal 120 bp was about 83%, while it was about 56% between TRAS-3-1 and the TRAS1 consensus sequence. The 3'-tail region of the LINE and SINE family is conserved strongly in many species (Malik and Eickbush 1998
; Ogiwara et al. 1999
), since the enzymatic machinery of LINEs may recognize higher-order structures of the 3' tail of LINE and SINE. When the 3'-tail region of L1Bm, another major non-LTR retrotransposon of the silkworm, is compared with a dozen genomic copies (Ichimura, Mita, and Sugaya 1997
), the sequence similarity averages about 85%. The above observation that the sequence similarity in the 3'-tail region is higher within the TRAS3 group (83%) but lower between TRAS1 and TRAS3 (56%) suggests that each TRAS family may be recognized by its own retrotransposition machinery but not by others.
In most non-LTR retrotransposons, the 5'-terminal regions are not conserved or are sometimes truncated by incomplete reverse transcription. The sequence uniformity of genomic copies of TRAS and SART elements, especially at the 5'-terminal regions, is therefore an unusual structure compared with other retroelements. This sequence uniformity may be partly determined by selective pressure arising from unequal crossover and gene conversion, as in the R1 and R2 insertion of 28S rDNA. Eickbush and his group concluded that the recombinational forces that work for concerted evolution of the rRNA genes can themselves rapidly amplify and eliminate copies of R1 and R2, independent of their ability to retrotranspose (Jakubczak et al. 1992
). Although organisms rely on a telomerase, telomere-telomere recombination is also thought to proceed by gene conversion and results in a net increase in telomeric DNA. Thus, this kind of recombination in the telomere region may contribute to the structural uniformity of telomeric-repeat-associated retrotransposons.
Identification of TRAS-like Elements in Lepidoptera
To study the more detailed structure of TRAS-like elements from various insect species, we employed the CODEHOP strategy (Rose et al. 1998
) for PCR amplification of unknown targets related to multiply aligned protein sequences (see Materials and Methods). This method was applied in practice to detect the diverse reverse transcriptase-like genes in the human genome. In general, the RT domain of non-LTR retrotransposons consists of seven conserved regions (Xiong and Eickbush 1990
; Nakamura et al. 1997
). The EN domain in the N-terminal region of ORF2 is thought to be responsible for target digestion and is relatively conserved among many non-LTR retroelements. Using PCR, therefore, we tried to amplify TRAS-like elements in the regions from EN to RT domains. At the beginning of the experiment, we had sequence information for TRAS1, TRAS3, TRAS4, TRAS5, and TRAS6 (partial sequence) of the silkworm. Based on the sequence comparison between these TRAS elements and other non-LTR elements, we designed a primer set within the conserved regions of EN and RT to amplify TRAS-like elements specifically but not other retroelements. Furthermore, to amplify the TRAS-like element from insects that are phylogenetically distant from the silkworm, we used two CODE hybrid oligonucleotide primers, CH-VVGI and CH-FADD (see Materials and Methods). They consisted of a short 3'-degenerate core region and a longer 5' consensus clamp region.
Southern hybridization with the Bombyx TRAS1 sequence as a probe suggested that TRAS-like elements were present in a variety of insect groups (data not shown). Thus, we tried several times but failed to detect the TRAS-like elements from insects outside Lepidoptera by the CODEHOP method. This means that the regions designed for PCR primers in these insects may have changed. However, we succeeded in isolating the TRAS-like sequences from two Lepidoptera, S. cynthia ricini and D. japonica. We isolated seven clones from S. cynthia ricini, which were divided into three families, TRASSC3, TRASSC4, and TRASSC9, based on sequence comparison. From D. japonica, we isolated one clone, named TRASDJ. However, we did not determine the 5'- and 3'-terminal sequences of the TRAS-like elements in S. cynthia and D. japonica, and therefore it is uncertain whether these retrotransposons are present in telomeric repeats.
Furthermore, we isolated EN-RT regions for two additional families, TRAS5 and TRAS6, from the silkworm, B. mori. However, we do not yet know the relationship between TRAS5 and TRAS6 and TRASY, TRASZ, and TRASW that were identified from lambda clones, since the structures of the latter groups were analyzed only in junction regions (see fig. 3 ).
The sequence comparison among TRAS elements from Bombyx and from two Lepidoptera revealed several features. The EN to RT regions of B. mori TRAS elements are highly conserved (50%72% in their amino acid sequences and 56%70% in their nucleic acid sequences). The amino acid identity between the Bombyx TRAS elements and R1Bm is only 25%27%, probably reflecting structural differences in functional domains, such as the region involved in sequence-specific digestion. Similar results were also obtained in a comparison of amino acid sequences among TRAS elements of Lepidoptera (37%55% amino acid identities within TRAS groups, 25%30% among TRAS and R1Bm). As far as we know, R1 is phylogenetically the most closely related element to TRAS. Therefore, the higher amino acid sequence identity within TRAS groups (about 37%70%) than among TRAS elements and R1 (about 25%30%) suggests that putative TRAS-like elements identified here can be classified into the same group. The phylogenetic tree constructed based on amino acid sequence from EN to RT supports the above idea. As shown in figure 4 , all TRAS elements isolated from three Lepidoptera constitute a single phylogenetic group and are closely related to other site-specific elements, R1Bm, RT1Ag, and SART1.
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Strict target specificity may also be ensured by another region in addition to the EN domain, which might be required for recognizing the longer arrays of telomeric repeats or telomeres. To search the TTAGG recognition domain in the TRAS, it is interesting that human telomeric repeat binding factor hTRF1 can bind to (TTAGGG)2 with its Myb domain (König, Fairall, and Rhodes 1998
). In addition, a recent report of Eickbush's group suggested that a site-specific retroelement, R2, also retained the Myb-like domain near the N-terminal region of the ORF (Burke et al. 1999
). The Myb domain is usually composed of 5060 amino acids forming three helices and is found in many DNA-binding proteins, such as the MYB oncogene (Ogata et al. 1994
) and Engrailed (Ades and Sauer 1994
). To determine whether the TTAGG-specific retrotransposon TRAS also has the Myb-like domain, we searched the helix structure from EN to RT domains using a secondary-structure prediction program (Hitachi, DNASIS). Consequently, we found a helix-turn-helix motif between the EN and RT domains not only in all TRAS elements, but also in other non-LTR retrotransposons, R1Bm, SART1, RT1Ag, TARTDm, and L1Hs (data not shown).
In the Myb-related proteins so far reported, amino acid residues themselves are not conserved strictly in three helices, but the specific positions that are occupied by hydrophobic or charged amino acids are conserved (König and Rhodes 1997
). To know whether these charged and hydrophobic residues are conserved within the putative helix-turn-helix regions predicted above, we compared amino acid sequences in corresponding regions of TRAS and other retrotransposons (data not shown). We found that TRAS elements have highly conserved amino acids with known Myb domains (fig. 6
), while these residues were changed at several sites in retrotransposons in addition to TRAS. Therefore, we speculate that highly conserved regions of TRAS between EN and RT may be responsible for DNA binding through a putative Myb-like function. There is a possibility that some cysteine-histidine motifs found in both ORF1 and ORF2, or this Myb-like domain, may be required for recognizing the longer arrays of telomeric repeats or telomeres. Further studies, such as DNA-binding analysis, may clarify the above hypothesis.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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3 Abbreviations: EN, endonuclease; EST, expression sequence tag; non-LTR, nonlong terminal repeat; ORF, open reading frame; PCR, polymerase chain reaction; RT, reverse transcriptase.
1 Keywords: site-specific non-LTR retrotransposon
TRAS family
telomeric repeat
Bombyx mori
evolution
Myb-like domain
2 Address for correspondence and reprints: Haruhiko Fujiwara, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. haruh{at}k.u-tokyo.ac.jp
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