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MBE Advance Access originally published online on November 17, 2004
Molecular Biology and Evolution 2005 22(3):673-682; doi:10.1093/molbev/msi054
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Molecular Biology and Evolution vol. 22 no. 3 © Society for Molecular Biology and Evolution 2004; all rights reserved.

Research Article

Isolation and Characterization of Active LINE and SINEs from the Eel

Masaki Kajikawa, Kenji Ichiyanagi, Nozomu Tanaka and Norihiro Okada

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Kanagawa 226-8501, Japan

E-mail: nokada{at}bio.titech.ac.jp.


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Long interspersed elements (LINEs) and short interspersed elements (SINEs) are retrotransposons. These elements can mobilize by the "copy-and-paste" mechanism, in which their own RNA is reverse-transcribed into complementary DNA (cDNA). LINEs and SINEs not only are components of eukaryotic genomes but also drivers of genomic evolution. Thus, studies of the amplification mechanism of LINEs and SINEs are important for understanding eukaryotic genome evolution. Here we report the characterization of one LINE family (UnaL2) and two SINE families (UnaSINE1 and UnaSINE2) from the eel (Anguilla japonica) genome. UnaL2 is ~3.6 kilobases (kb) and encodes only one open reading frame (ORF). UnaL2 belongs to the stringent type—thought to be a major group of LINEs—and can mobilize in HeLa cells. We also show that UnaL2 and the two UnaSINEs have similar 3' tails, and that both UnaSINE1 and UnaSINE2 can be mobilized by UnaL2 in HeLa cells. These elements are thus useful for delineating the amplification mechanism of stringent type LINEs as well as that of SINEs.

Key Words: transposable element • retrotransposon • retrotransposition • reverse transcriptase • endonuclease


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Long interspersed elements (LINEs) and Short interspersed elements (SINEs) are transposable elements in eukaryotic genomes that mobilize through an RNA intermediate. These elements are first transcribed into RNA, and the RNA is then reverse-transcribed into cDNA that is subsequently integrated at a new location within the host genome. This "copy-and-paste" mechanism is called retrotransposition, a process that expands the number of LINEs and SINEs such that they often occupy a considerable portion of a eukaryotic genome. For example, the human genome includes ~850,000 copies of LINEs and ~1,500,000 copies of SINEs, and ~34% of the genome is composed of LINEs and SINEs (Lander et al. 2001). Hence LINEs and SINEs are a large component of eukaryotic genomes. These elements are also thought to have a significant impact on the evolution and complexity of eukaryotic genomes (Kazazian 2004). Thus, understanding their amplification mechanism is indispensable to understanding the evolution of eukaryotic genomes.

LINEs are ~4-7 kb and comprise a 5' untranslated region (UTR), open reading frames (ORFs), and a 3' UTR. LINEs usually encode two ORFs. ORF1 encodes a nucleic acid–binding protein that can bind its own RNA (Hohjoh and Singer 1997; Kolosha and Martin 1997) and is thought to form a retrotransposition intermediate. In addition, it has been reported that this protein also possesses a nucleic acid chaperone activity (Martin and Bushman 2001), although its role in retrotransposition processes is unclear. ORF2 encodes an endonuclease (EN; Feng et al. 1996) and a reverse transcriptase (RT; Xiong and Eickbush 1990). Through phylogenetic analysis of LINE RTs, LINEs can be divided into 12 families or clades (Malik, Burke, and Eickbush 1999; Lovsin, Gubensek, and Kordis 2001; Ogiwara et al. 2002).

A biochemical analysis of the amplification mechanism of LINEs was first performed using the silkworm LINE R2Bm (Luan et al. 1993; Luan and Eickbush 1995; Yang and Eickbush 1998). By the analysis it is revealed that the LINE EN nicks a target site in the host genome, and the LINE RT initiates reverse transcription of its own RNA from the 3' hydroxyl group that is generated by the nick (Luan et al. 1993). This reaction, in which the cleavage and the reverse transcription occur simultaneously, is called target-primed reverse transcription (TPRT). Besides R2Bm, the amplification mechanism of the human L1 has also been well studied via a retrotransposition assay in mammalian cells (mainly in HeLa cells; e.g., Moran et al. 1996). L1 elements retrotranspose at high frequency in HeLa cells (Moran et al. 1996), and L1 ORF1 and ORF2 (EN and RT) are required for L1 retrotransposition (Feng et al. 1996; Moran et al. 1996). Perhaps 80–100 copies of retrotransposition-competent L1 are present in the human genome (Brouha et al. 2003), and L1 can mobilize in trans and generate processed pseudogenes (Esnault, Maestre, and Heidmann 2000; Wei et al. 2001). L1 can also mobilize human SINE; Alu in trans (Dewannieux, Esnault, and Heidmann 2003). L1s, along with sequences derived from their 3' flanking regions, retrotranspose to new locations (3' transduction; Moran, DeBerardinis, and Kazazian 1999). Furthermore, a large-scale deletion or inversion can be generated during L1 retrotransposition (Gilbert, Lutz-Prigge, and Moran 2002; Symer et al. 2002). These data provide convincing evidence for detailed mechanisms of mammalian genome diversification. One intriguing discovery of L1 elements is that, except for the poly(A) tail, there is apparently no strict sequence requirement in the 3' tail for recognition by RT during retrotransposition (Moran et al. 1996).

SINEs are ~100-500 bp long. They are composed of a tRNA-related region and a tRNA-unrelated region, except for the human SINE Alu, which has a 7SL RNA-related region instead of the tRNA-related region. SINEs do not encode any protein required for retrotransposition, so SINEs must recruit the enzymatic machinery for their own retrotransposition and are thus non-autonomous transposable elements. We previously reported that there are several LINE/SINE pairs that share a similar 3' tail in eukaryotic genomes (Okada et al. 1997). These pairs are distributed in several clades, such as the LINE2 (L2) clade (Smit 1996; Terai, Takahashi, and Okada 1998; Ogiwara et al. 2002), the CR1 clade (Ohshima et al. 1996; Kajikawa, Ohshima, and Okada, 1997; Ogiwara et al. 1999; Ogiwara et al. 2002), the RTE clade (Okada and Hamada 1997), and the Tad1 clade (Okada et al. 1997). Based on the TPRT reaction, the initiation of reverse transcription occurs at the 3' end of LINEs. Hence we proposed that these SINEs are mobilized by their partner LINEs (Ohshima et al. 1996; Okada et al. 1997). The RT of silkworm LINE R2Bm specifically recognizes the 3' UTR of its own RNA for the initiation of reverse transcription (Luan and Eickbush 1995; Mathews et al. 1997), although a partner SINE that shares the same 3' tail with R2Bm has not been discovered. We therefore proposed that LINEs can be divided in two groups, the stringent type and the relaxed type, based on 3' tail recognition (Okada et al. 1997). The stringent type strictly recognizes its own 3' tail for retrotransposition, whereas the relaxed type does not require a specific sequence at the 3' tail, except for the poly(A), for retrotransposition. At present, human L1 is the only example of the relaxed type, so it is thought that the majority of LINEs are of the stringent type.

Here we use the retrotransposition assay in a cultured cell line to analyze the amplification mechanism of LINEs (and SINEs) of the stringent type. We previously demonstrated that UnaL2, a stringent LINE from the eel, can retrotranspose in HeLa cells and that UnaSINE1, which has a similar 3' tail, can retrotranspose using the enzymatic machinery of UnaL2 (Kajikawa and Okada 2002). We have now isolated a second SINE from the eel genome, UnaSINE2, and report here the further characterization of the eel LINE and SINEs.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Isolation of UnaL2 and UnaSINEs
A genomic library was constructed from eel (Anguilla japonica) to identify the genomic DNA sequence of the LINE element. A LINE-like element in the eel genome was partially characterized by Ohshima et al. (1996). We determined the entire UnaL2 sequence using genomic DNA walking (Ohshima et al. 1996; Kajikawa, Ohshima, and Okada 1997). A screen of the eel genomic library yielded eight phage clones. Using primers UnFw-6 and UnRv-16, each of which was designed to anneal to the distal end of UnaL2, UnaL2 sequences were amplified by PCR from phage DNAs and were directly determined by cycle sequencing. Three of eight UnaL2 sequences contained a highly conserved ORF (amino acid identity >99.5%). UnaL2 from the Aja6-15 phage clone was also highly conserved, having only two amino acid replacements as compared with the consensus sequence of the three UnaL2s that contained the highly conserved ORF. To isolate SINE sequences from the eel genomic library, we used in vitro–labeled transcripts from eel total genomic DNA as probes (Endoh and Okada 1986). Positive phage clones were isolated, and their inserts were subcloned into the plasmid pUC18. Inserts were sequenced with the M4 or RV primer, and seven UnaSINE1s and four UnaSINE2s were characterized.

Estimation of the Copy Number of UnaL2 and UnaSINEs
For dot blot analyses, progressively decreasing amounts of genomic DNA from A. japonica and cloned DNA were dotted on a membrane. Polymerase chain reaction (PCR) products of ~150 bp that corresponded to sequences from UnaL2, UnaSINE1, or UnaSINE2 were labeled by BcaBEST DNA polymerase (TaKaRa) using [{alpha}-32P]dCTP. Hybridization was performed at 42°C in 50% formamide. Washing was performed in 2x SSC and 1% sodium dodecyl sulfate (SDS) at 55°C for 30 min. From comparisons of the intensities of spots obtained with the genomic DNA and the cloned DNA, we estimated the element copy number. The haploid genome of the eel was assumed to contain 1 x 109 bp.

Retrotransposition Assay
Plasmids used for the cis and trans retrotransposition assay (Kajikawa and Okada 2002) were purified by the QIAfilter Plasmid Midi kit (Qiagen) according to the manufacturer's instructions. Complete experimental procedures are available on request.

HeLa cells were grown in high-glucose Dulbecco's modified Eagle's medium (Invitrogen) in the absence of pyruvate and supplemented with 2 mM L-glutamine and 10% fetal bovine serum. All cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2. For the assay, HeLa cells (2 x 105 cells/well) were seeded in six-well plates and transfected with 1 µg plasmid DNA with 3 µl of FuGENE6 reagent (Roche) according to the manufacturer's instructions. For co-transfection, we used 6 µl of FuGENE6 reagent with 1 µg of each plasmid DNA (2 µg DNA total). After transfection, HygR cells were selected with 200 µg/ml hygromycin. In co-transfections, the selection was performed with 200 µg/ml hygromycin and 15 mM L-histidinol. By comparing the data with cell survival results from negative controls, we estimated that ≥95% of transfected cells became antibiotic resistant (HygR cells or HygR and HisR cells). The antibiotic-resistant cells were then trypsinized and seeded to a density of ~2 x 105 to 4 x 106 cells per 100-mm plate and grown in medium containing 400 µg/ml G-418. After G-418 selection, plates were fixed with 100% ethanol and stained with 2% Giemsa's solution. G-418R colonies were counted, and the retrotransposition frequencies were calculated as the number of G-418R colonies per one plated cell with HygR, or one plated cell with HygR and HisR.

Accession Numbers
The UnaL2 nucleotide sequence of the clone Aja 6-15 has been submitted to the DNA Databank of Japan (DDBJ) and assigned the accession number AB179624. Nucleotide sequences of seven UnaSINE1s and four UnaSINE2s have been submitted to DDBJ and assigned accession numbers AB179625, AB179626, AB179627, AB179628, AB179629, AB179630, and AB179631 for the UnaSINE1s; accession numbers AB179632, AB179633, AB179634, and AB179635 for the UnaSINE2s.


   Results and Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Conclusions
 Acknowledgements
 References
 
Isolation of LINEs from the Eel Genome
Using genomic walking of the eel genome, we determined a consensus sequence of a full-length eel LINE and named this LINE UnaL2 (from unagi, Japanese for "eel"). The full-length consensus sequence of UnaL2 is approximately 3.6 kb and encodes one ORF (fig. 1A). Using dot blot hybridization, we estimated the copy number of UnaL2 per haploid eel genome to be ~100 when using a 5' probe and ~350 when using a probe that was 1 kb from the 3' end of UnaL2. Thus UnaL2 has various 5' truncations, as do other LINEs. A database search using UnaL2 revealed that a highly homologous LINE exists in the zebrafish genome. Both UnaL2 and the zebrafish LINE encode only one ORF, which corresponds to ORF2 of other LINEs (fig. 1A). Amino acid sequences of the proteins encoded by UnaL2 and the zebrafish LINE are ~55% identical. Based on the phylogenetic analysis of RT sequences, UnaL2 and the zebrafish LINE belong to the L2 clade. Truncated sequences of the zebrafish LINE were originally reported by our group, and we called this LINE ZfL2 (for zebrafish; Okada et al. 1997; Ogiwara et al. 2002). Later, Jurka's group deduced the entire ZfL2 consensus sequence using sequences of the zebrafish genome database and called this LINE CR1-2_DR (Kapitonov and Jurka 2003). Here we call this LINE ZfL2.



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  		FIG. 1.— The structure of UnaL2 and ZfL2. (A) Schematic representation of UnaL2 and ZfL2. UnaL2 and ZfL2 are composed of the 5' UTR, the ORF, and the 3' UTR. The horizontal arrowheads indicate repeat units of the repeat sequence in the 5' UTR. Shaded boxes indicate the ORFs. EN, endonuclease. RT, reverse transcriptase. (B) The repeat sequence in the 5' UTR of UnaL2 (Aja6-15). The repeat unit is indicated by an underline and dashed underline (see Results and Discussion). Numbers indicate the distance from the 5' end of UnaL2. (C) The 3' conserved region of UnaL2 and ZfL2. The 3'-terminal region of UnaL2 and ZfL2 is well conserved. UnaL2, upper sequence (this study, Aja6-15); ZfL2 (CR1-2_DR), lower sequence (Kapitonov 2002). The stem region is indicated by dashed underlines. The putative poly(A) signal is underlined by a single line. The 3'-terminal repeat is indicated by double underlines. (D) Putative secondary structure of the 3' conserved region of UnaL2 RNA.

 
We isolated and sequenced eight full-length UnaL2s from an eel genomic library. Three of the eight were very similar (Aja6-5, Aja6-15, and Aja6-18). Each was 99.5%–99.9% identical to the consensus nucleotide sequence of the three UnaL2s, suggesting that these UnaL2s were amplified very recently and that UnaL2 is retrotransposition competent in the eel genome. Then, as described elsewhere (Kajikawa and Okada 2002), we performed a retrotransposition assay in HeLa cells using UnaL2 (Aja6-15) and showed that UnaL2 can retrotranspose in HeLa cells.

Characterization of the 5' UTR of UnaL2
The 5' UTR of UnaL2 is ~400 bp and includes a series of 39-bp repeats (fig. 1B, underlined sequence) that are separated by an internal sequence of 24 bp. In some UnaL2s, this 63-bp unit (dashed underline) is also repeated in tandem, with the number of repeats varying in different UnaL2s (one to several units). In comparison, the 5' UTR of ZfL2 is ~1.1 kb, most of which is composed of a tandem repeat of an ~300-bp unit (Kapitonov 2002). Repeat sequences occur in 5' UTRs of other LINEs, but the length and sequence of the repeats vary (Kajikawa, Ohshima, and Okada 1997; for a review on mouse L1s, see Moran and Gilbert 2002). It is thought that LINEs have an internal promoter in the 5' UTR, so the repeat sequences may be important for promoter activity. In the case of the mouse L1 LINE subfamily, TF, the 5' UTR contains a tandem repeat of a monomer unit (~200 bp), and the promoter activity of mouse L1 is proportional to the number of monomers (DeBerardinis and Kazazian 1999). Hence, the repeat sequences of UnaL2 might also have promoter activity.

Characterization of the 3' UTR of UnaL2
The 3' UTR of UnaL2 is ~330 bp, and although it is not well conserved with the 230-bp ZfL2 3' UTR, their 3'-terminal regions are well conserved (fig. 1C). This suggests that the 3' tails of UnaL2 and ZfL2 are important for retrotransposition. The conserved tail is composed of two parts, the stem-loop region and the terminal repeat region (figs. 1C and D; Kajikawa and Okada 2002). Both regions of UnaL2 are required for retrotransposition (Kajikawa and Okada 2002). The stem-loop region is thought to function as a recognition site for the UnaL2 protein (UnaL2p) when this region is transcribed in the RNA (Baba et al. 2004). The terminal repeat region of UnaL2 is probably required for the slippage reaction during reverse transcription initiation (Kajikawa and Okada 2002). These data indicate that UnaL2 and ZfL2 belong to the stringent type of LINEs. The 3' tail of UnaL2 contains a putative poly(A) signal (fig. 1C, underline), whereas ZfL2 does not. Thus, the poly(A) signal in these LINEs may be dispensable for retrotransposition, and the poly(A) signal of the host genome located downstream of the LINE might lead to poly(A) addition to the RNA of these LINEs.

Implication from Structural Modeling of UnaL2
The amino acid sequence of the N-terminal region (1–250) of UnaL2p shows similarity to members of the apurine/apyrimidine (AP) endonuclease family (figs. 1A and 2A), most of which possess an activity specific to AP sites in duplex DNA. The co-crystal structure of the AP endonuclease of hHAP1 with substrate DNA has revealed that AP site recognition uses two protruding elements (AP-pinch1 and AP-pinch2; figs. 2A and 2B), which together pinch the AP site duplex from both sides of the helix (Mol et al. 2000). Moreover, Phe-266, Trp-280, and Leu-282 (fig. 2A, triangles) comprise a pocket for binding of the "flipped-out" abasic deoxyribose. A sequence alignment based on 3D–1D profiles made by the program 3D-PSSM (Fischer et al. 1999) suggests that UnaL2p lacks these elements and residues. Using this alignment, we modeled a 3D structure of the UnaL2p endonuclease domain using the program MODELLER (Sali and Blundell 1993; fig. 2B). Although the structure of the endonuclease core is very similar to that of hHAP1, the modeled structure does not possess the AP-pinch regions. Moreover, UnaL2p lacks the pocket that packs the flipped-out abasic deoxyribose. Weichenrieder, Repanas, and Perrakis (2004) recently resolved the structure of the L1Hs endonuclease and suggested that Phe-193, Ser-202, and Ile-204 (which correspond to Phe-266, Trp-280, and Leu-282 of hHAP1) accommodate an extrahelical adenosine next to the scissile bond. Because its substrate (5'TTTT-AA3', hyphen indicates cleavage) is unusual, as the adenosine next to the scissile bond tends to be flipped out, it is still pending if LINE endonucleases generally recognize an extrahelical nucleotide. The lack of a corresponding pocket in UnaL2p (and Tad endonuclease) argues against the generality of the extrahelical nucleotide recognition. In addition, the sequence alignment suggests the absence of the AP-pinch regions in many LINE endonucleases, which may explain the fact that they are not specific to AP sites.



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  		FIG. 2.— LINE-encoded endonucleases. (A) Sequence alignment of LINE-encoded endonucleases with AP endonucleases. The alignments are based on 3D-1D profiles made by 3D-PSSM. The secondary structures (hHap1, Xth(Ec), and L1Hs, observed; others, predicted) are in red and light blue for helices and strands, respectively. AP pinch regions in hHAP1 that recognize AP site DNA are boxed. Residues in hHAP1 and L1Hs that pack, or are proposed to pack an extrahelical nucleotide are indicated by closed triangles. hHap1, human HAP1; Xth(Ec), Escherichia coli Xth; Xth(Ct), Chlorobium tepidum Xth. Sources of sequences are as follows (notations refer to accession numbers unless otherwise indicated): hHAP1, CAA42437; Xth(Ec), BAA15544; Xth(Ct), AAM73352; UnaL2, this study (Aja6-15); Jockey, AAA28675 [GenBank] L1Tc, CAB41692 [GenBank] LOA, Felger and Hunt (1992); TRAS, BAB21511 [GenBank] Tad, AAA21792 [GenBank] L1Hs, AAB59368 [GenBank] (B) A modeled structure of the endonuclease domain of UnaL2p. A homology-based 3D model was built on MODELLER using hHAP1 (pdb code:1BIX) as a template. AP pinch regions in hHAP1 are indicated in green. The ribbon models were drawn on PyMOL (DeLano 2002).

 
The Carboxyl-Terminal Conserved Region (CTCR) of UnaL2p is Required for Retrotransposition
The extreme carboxyl-terminal end of ORF2 proteins encoded by the L2 clade LINEs are well conserved (fig. 3A). This conserved region is also found in proteins of the CR1 clade. The CTCR of the CR1 clade is a part of the region B previously reported as a conserved region among this clade (Drew and Brindley 1997). Although well conserved, it was not known if the CTCR is required for retrotransposition. Thus we performed a retrotransposition assay of UnaL2s that have a point mutation in the CTCR (fig. 3). Mutations in the conserved residues of the CTCR abolished UnaL2 retrotransposition, whereas mutations in nonconserved residues did not influence UnaL2 retrotransposition frequency. These results suggest that the CTCR is the functional domain for retrotransposition of UnaL2. We hypothesize that the CTCR is used for 3' tail recognition because LINEs of the L2 and the CR1 clades are thought to recognize a stem-loop of their own RNA for reverse transcription (Ogiwara et al. 1999; Kajikawa and Okada 2002). If this hypothesis is valid, LINEs of other clades that also belong to the stringent type might have a different domain for the recognition of their own 3' tail.



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  		FIG. 3.— The carboxyl-terminal conserved region (CTCR) of UnaL2 is required for retrotransposition. (A) Alignment of the carboxyl-terminal region. Upper and lower four LINEs in the alignment belong to the L2 clade and the CR1 clade, respectively. Numbers above the alignment indicate the number of RT domains. Boxes indicate amino acids that are conserved in all LINEs of each clade. Residues conserved in all eight LINEs are highlighted by shaded boxes. Stop codons are indicated by asterisks. Sources of sequences are as follows (notations refer to accession numbers unless otherwise indicated): UnaL2, this study (Aja6-15); ZfL2 (CR1-2_DR), Kapitonov (2002); Human L2, Repbase Update at www.girinst.org/Repbase_Update; CR1-1_DR, Kapitonov and Jurka (2002a); CR1, AAC60281 [GenBank] PsCR1, BAA88337 [GenBank] HER1, Ogiwara et al. (1999); SmRT1, AAC06263 [GenBank] (B) Retrotransposition assay of UnaL2 mutants that have a point mutation in the CTCR. Images show G-418R cell colonies on each 100-mm plate selected from approximately 2–4 x 106 HygR cells. At least four independent experiments were done for each construct. RF, retrotransposition frequency.

 
Isolation of SINEs from the Eel Genome
We screened an eel genomic library to isolate an eel SINE using an RNA probe transcribed from eel genomic DNA. Two kinds of SINEs were isolated, UnaSINE1 and UnaSINE2. UnaSINE1 (~230 bp) comprises three parts, a tRNA-related region, an internal region, and a 3' conserved region, which ends with [TGTAA]n (n = ~3; Kajikawa and Okada 2002; fig. 4A). We isolated seven UnaSINE1 clones, and the divergence of these clones from the consensus sequence, namely the average sequence divergence, varied from ~1% to 15% (data not shown). From dot blot hybridization, we estimated that the UnaSINE1 copy number is ~1000–6000 per haploid eel genome. UnaSINE2 (~200 bp) also comprises three parts, but the 3'-terminal repeat is [CCATTTA]n (n = 2–3). We isolated four different UnaSINE2 clones, which were all highly identical (>98%) to the consensus sequence. The estimated copy number of UnaSINE2 is 1500–3000 per haploid eel genome. These two SINEs are similar to the salmon SINE called SmaI (fig. 4A; Kido et al. 1991).



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  		FIG. 4.— Alignment of UnaSINEs. (A) Alignment of UnaSINE1, UnaSINE2, SmaI SINE, and Lys tRNA. Box A and box B indicate the internal promoter; region a, the tRNA-related region; region b, the internal region, which is similar to the core sequence; region c, the 3' conserved region. The stem regions are indicated by dashed underlines. The 3'-terminal repeats are underlined. Numbers indicate the distance from the 5' ends. Sources of sequences are as follows (notations refer to accession numbers unless otherwise indicated): Lys tRNA (rabbit Lys tRNA), K00289 [GenBank] ; SmaI (Kido et al. [1991]; consensus sequence); UnaSINE1, this study (consensus sequence); UnaSINE2, this study (consensus sequence); UnaL2, this study (Aja6-15). (B) The alignment of the core regions. CORE (Ther-1), the core sequence described by Gilbert and Labuda (1999).

 
Characterization of the 3'-Terminal Region of the Eel SINEs
UnaSINE1, UnaSINE2, and SmaI SINE have a similar 36-bp 3' region that corresponds to the stem-loop region of UnaL2 (fig. 4A, region c). Each of the three SINEs ends with a unique 3'-terminal repeat: [TGTAA] for UnaSINE1, [CCATTTA] for UnaSINE2, and [ATT] for SmaI SINE. We have shown that UnaSINE1 is mobilized by the enzymatic machinery of UnaL2 (Kajikawa and Okada 2002). The 3' tail of UnaSINE2 is also similar to that of UnaL2, but the sequence just downstream of the stem and the 3'-terminal repeat sequence differs from those of UnaL2 (fig. 4A). In a trans retrotransposition assay (Kajikawa and Okada 2002), UnaSINE2 was also mobilized by the enzymatic machinery of UnaL2 (fig. 5). Hence the sequence just downstream of the stem appears not to be important for retrotransposition. The sequence length between the stem and the 3'-terminal repeat is, however, similar among these SINEs and LINEs and thus might be important for retrotransposition. The 3'-terminal repeat region is the initiation site of reverse transcription, and the stem-loop region is probably the recognition site for UnaL2p (Baba et al. 2004). UnaL2p might determine the initiation site by measuring the distance from the stem. The retrotransposition assay also indicates that [CCATTTA]n of UnaSINE2 is functional for retrotransposition. This is consistent with previous data that the repetition of the 3'-terminal repeat is important for retrotransposition and is probably required for the slippage reaction during reverse transcription initiation (Kajikawa and Okada 2002).



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  		FIG. 5.— trans retrotransposition assay. (A) Schematic of the trans retrotransposition assay in HeLa cells using the mneol cassette. When a test sequence (Tsq.) is recognized by the UnaL2 protein in trans and the mneol cassette is retrotransposed, HeLa cells become G-418 resistant. Neo, neomycin resistance gene. PCMV, cytomegalovirus promoter. PRSV, Rous sarcoma virus promoter. SVpA, SV40 poly(A) signal. (B) Results of the trans retrotransposition assay. N, the number of independent transfection experiments. RF, retrotransposition frequency.

 
The Core Sequence of the Eel SINEs
The largest difference between the eel SINEs and the salmon SINE is the presence or absence, respectively, of the internal region (fig. 4A). Although internal regions of UnaSINE1 and UnaSINE2 do not seem to be homologous, they have weak homology to the core (fig. 4A, region b, and fig. 4B). The core was reported as a conserved sequence among some SINEs isolated from highly diverged organisms (Gilbert and Labuda 1999). A role for the core sequence has not, however, been elucidated. The retrotransposition frequency of the full-length UnaSINE1 is about two times higher than that using only its 3' conserved region (fig. 5B). It seems that the core region of UnaSINE1 enhances retrotransposition. However, the retrotransposition frequency of full-length UnaSINE2 is lower than that using only its 3' conserved region. Thus, we do not know whether the core sequence really enhances the retrotransposition of UnaSINEs. The SmaI SINE dose not have the core sequence, and therefore the core may not be essential for retrotransposition. The reason the core sequence is conserved among diverse SINEs remains to be elucidated. UnaSINE1 and UnaSINE2 are transcribed by RNA polymerase II in this assay, but SINEs are transcribed by RNA polymerase III in vivo, so the core sequence might function (e.g., enhance retrotransposition) when these SINEs are transcribed by RNA polymerase III. A retrotransposition assay system in which the SINE sequence is transcribed by RNA polymerase III has been developed by Heidmann's group (Dewannieux, Esnault, and Heidmann 2003). With this system, the core function might be revealed. Alternatively, the core might have a host-related function and may not be required for retrotransposition.

The Stem-Loop of the 3' Tail is Conserved Among LINEs and SINEs of the L2 Clade
The 3' tails of UnaL2 and UnaSINEs are conserved and required for retrotransposition (figs. 4A and 5). The RNA transcribed from the conserved tail is proposed to form a stem-loop (Fig. 1D; Kajikawa and Okada 2002). Most LINEs (and SINEs) belonging to the L2 clade also have a 3' tail that purportedly forms a stem-loop similar to that of UnaL2. Alignment of the 3' tails from the L2 clade LINEs and SINEs is shown in figure 6. All of the L2 clade LINEs end with a repeat sequence, although the nucleotide sequence of the repeats differs among the members. The stem region (underlined) is well conserved, whereas the loop region varies in length and sequence. The sequences that are 5' and 3' to the stem are also well conserved (dashed underlines). This conservation suggests that LINE proteins may use a common mechanism for recognizing the 3' tails of LINE RNAs. The retrotransposition machinery of UnaL2 can recognize the 3' tail of UnaSINE1 and UnaSINE2 as well as its own 3' tail, but it cannot recognize those of CiLINE2 and Af1SINE (Kajikawa and Okada 2002). These tails differ substantially in the loop region. Thus, we speculate that the stem is used as a common binding site for LINE proteins, and the loop region is used for LINE-specific recognition (Baba et al. 2004).



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  		FIG. 6.— Alignment of the 3'-terminal tails of LINEs and SINEs belonging to the L2 clade. Boxed names refer to LINEs and others refer to SINEs. The 3'-terminal repeats are indicated by bracketed nucleotides. The stem regions are underlined. Conserved nucleotides other than the stem are indicated by dashed underlines. Vertical lines in the stem indicate an additional nucleotide: CR1-1_DR, T; CR1-3_DR, T; CR1-4_DR, A; SINE3-1, T. Sources of sequences are as follows (notations refer to accession numbers unless otherwise indicated): UnaL2, this study (Aja6-15); ZfL2 (CR1-2_DR), Kapitonov (2002); UnaSINE1, this study (consensus sequence); UnaSINE2, this study (consensus sequence); SmaI (Kido et al. [1991]; consensus sequence); Human L2 and MIR1, Repbase Update at www.girinst.org/Repbase_Update; CR1-1_DR, Kapitonov and Jurka (2002a); CR1-3_DR, Kapitonov and Jurka (2002b); CR1-4_DR, Kapitonov and Jurka (2002c); SINE3-1, Kapitonov and Jurka (2002d); CiLINE2 and Af1SINE, Terai, Takahashi and Okada [1998]; consensus sequence); Maui, AF086712 [GenBank] .

 

   Conclusions
TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
Acknowledgements
 References
 
We have isolated one LINE and two SINEs from the eel genome (UnaL2, UnaSINE1, and UnaSINE2, respectively). UnaL2 can retrotranspose in HeLa cells and can mobilize UnaSINE1 and UnaSINE2. UnaL2 belongs to the L2 clade and the stringent type of LINEs that strictly recognize their own 3' tail for retrotransposition. Hence UnaL2 and UnaSINEs will be useful for studying the amplification mechanism of stringent-type LINEs (and SINEs) using retrotransposition assays in HeLa cells. These assays should reveal the influences of the stringent-type LINEs (and SINEs) on the evolution of the eukaryotic genome as they further our understanding of the amplification mechanism of this type of element.


   Acknowledgements
TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
References
 
We thank Drs. M. Ohta and S. Fukai (Tokyo Inst. Tech.) for assisting with homology modeling and preparing ribbon models, respectively. This work was supported by a Grant-in-Aid to N.O. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Billie Swalla, Associate Editor


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 

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Accepted for publication November 8, 2004.


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