Kenji K Kojima

Abstract: Autonomous non-long terminal repeat (non-LTR) retrotransposons and their repetitive remnants are ubiquitous components of mammalian genomes. Recently, we identified non-LTR retrotransposon families, Ingi-1_AAl and Ingi-1_EE, in two hedgehog genomes. Here we rename them to Vingi-1_AAl and Vingi-1_EE and report a new clade "Vingi", which is a sister clade of Ingi that lacks the ribonuclease H domain. In the European hedgehog genome, there are 11 non-autonomous families of elements derived from Vingi-1_EE by internal deletions. No retrotransposons related to Vingi elements were found in any of the remaining 33 mammalian genomes nearly completely sequenced to date, but we identified several new families of Vingi and Ingi retrotransposons outside mammals. Our data suggest the horizontal transfer of Vingi elements to hedgehog, although the vertical transfer cannot be ruled out. The compact structure and trans-mobilization of non-autonomous derivatives of Vingi can make them useful for in vivo retrotransposition assay system.

Abstract: Alu is a predominant short interspersed element (SINE) family in the human genome and consists of two monomer units connected by an A-rich linker. At present, dimeric Alu elements are active in humans, but Alu monomers are present as fossilized sequences. A comparative genome analysis of human and chimpanzee genomes revealed 8 recent insertions of Alu monomers. One of them was a retroposed product of another Alu monomer with 3' transduction. Further analysis of 1404 loci of the Alu monomer in the human genome revealed that some Alu monomers were recently generated by recombination between the internal and 3' A-rich tracts inside of dimeric Alu elements. The data show that Alu monomers were generated by (1) retroposition of other Alu monomers, and (2) recombination between 2 A-rich tracts.

Abstract: ABSTRACT: BACKGROUND: Eukaryotic genomes harbor diverse families of repetitive DNA derived from transposable elements (TEs) that are able to replicate and insert into genomic DNA. The biological role of TEs remains unclear, although they have profound mutagenic impact on eukaryotic genomes and the origin of repetitive families often correlates with speciation events. We present a new hypothesis to explain the observed correlations based on classical concepts of population genetics. PRESENTATION OF THE HYPOTHESIS: The main thesis presented in this paper is that the TE-derived repetitive families originate primarily by genetic drift in small populations derived mostly by subdivisions of large populations into subpopulations. We outline the potential impact of the emerging repetitive families on genetic diversification of different subpopulations, and discuss implications of such diversification for the origin of new species. TESTING THE HYPOTHESIS: Several testable predictions of the hypothesis are examined. First, we focus on the prediction that the number of diverse families of TEs fixed in a representative genome of a particular species positively correlates with the cumulative number of subpopulations (demes) in the historical metapopulation from which the species has emerged. Furthermore, we present evidence indicating that human AluYa5 and AluYb8 families might have originated in separate proto-human subpopulations. We also revisit prior evidence linking the origin of repetitive families to mammalian phylogeny and present additional evidence linking repetitive families to speciation based on mammalian taxonomy. Finally, we discuss evidence that mammalian orders represented by the largest numbers of species may be subject to relatively recent population subdivisions and speciation events. IMPLICATIONS OF THE HYPOTHESIS: The hypothesis implies that subdivision of a population into small subpopulations is the major step in the origin of new families of TEs as well as of new species. The origin of new subpopulations is likely to be driven by the availability of new biological niches, consistent with the hypothesis of punctuated equilibria. The hypothesis also has implications for the ongoing debate on the role of genetic drift in genome evolution. Reviewers This article was reviewed by Eugene Koonin, Juergen Brosius and I. King Jordan.

Abstract: The wheat stripe rust fungus (Puccinia striiformis f. sp. tritici, PST) is responsible for significant yield losses in wheat production worldwide. In spite of its economic importance, the PST genomic sequence is not currently available. Fortunately Next Generation Sequencing (NGS) has radically improved sequencing speed and efficiency with a great reduction in costs compared to traditional sequencing technologies. We used Illumina sequencing to rapidly access the genomic sequence of the highly virulent PST race 130 (PST-130).

Abstract: ABSTRACT:Background "Domestication" of transposable elements (TEs) led to evolutionary breakthroughs such as the origin of telomerase and the vertebrate adaptive immune system. These breakthroughs were accomplished by the adaptation of molecular functions essential for TEs, such as reverse transcription, DNA cutting and ligation or DNA binding. Cryptons represent a unique class of DNA transposons using tyrosine recombinase (YR) to cut and rejoin the recombining DNA molecules. Cryptons were originally identified in fungi and later in the sea anemone, sea urchin and insects. Results Herein we report new Cryptons from animals, fungi, oomycetes and diatom, as well as widely conserved genes derived from ancient Crypton domestication events. Phylogenetic analysis based on the YR sequences supports four deep divisions of Crypton elements. We found that the domain of unknown function 3504 (DUF3504) in eukaryotes is derived from Crypton YR. DUF3504 is similar to YR but lacks most of the residues of the catalytic tetrad (R-H-R-Y). Genes containing the DUF3504 domain are potassium channel tetramerization domain containing 1 (KCTD1), KIAA1958, zinc finger MYM type 2 (ZMYM2), ZMYM3, ZMYM4, glutamine-rich protein 1 (QRICH1) and "without children" (WOC). The DUF3504 genes are highly conserved and are found in almost all jawed vertebrates. The sequence, domain structure, intron positions and synteny blocks support the view that ZMYM2, ZMYM3, ZMYM4, and possibly QRICH1, were derived from WOC through two rounds of genome duplication in early vertebrate evolution. WOC is observed widely among bilaterians. There could be four independent events of Crypton domestication, and one of them, generating WOC/ZMYM, predated the birth of bilaterian animals. This is the third-oldest domestication event known to date, following the domestication generating telomerase reverse transcriptase (TERT) and Prp8. Many Crypton-derived genes are transcriptional regulators with additional DNA-binding domains, and the acquisition of the DUF3504 domain could have added new regulatory pathways via protein-DNA or protein-protein interactions. Conclusions Cryptons have contributed to animal evolution through domestication of their YR sequences. The DUF3504 domains are domesticated YRs of animal Crypton elements.

Abstract: ABSTRACT: BACKGROUND: Long interspersed nuclear element-1 (LINE-1 or L1) is a dominant repetitive sequence in the human genome. Besides mediating its own retrotransposition, L1 can mobilize Alu and messenger RNA (mRNA) in trans, and probably also SVA and non-coding RNA. The structures of L1 copies and trans-mobilized retrocopies are variable and can be classified into three categories: full-length; 5'-truncated; and 5'-inverted insertions. These structures may be generated by different 5' integration mechanisms. RESULTS: In this study, a method to correctly characterize insertions with short target site duplications (TSDs) is developed and extranucleotides, TSDs and microhomologies (MHs) at junctions were analysed for the three types of insertions. Only 5'-truncated L1 insertions were found to be associated with short TSDs. Both full-length and 5'-truncated retrotransposed sequences in trans, including Alu, SVA and mRNA retrocopies and also full-length and 5'-inverted L1, were not associated with short TSDs, indicating the difference of 5' attachment between retrotransposition in cis and retrotransposition in trans. Target sequence analysis suggested that short TSDs were generated in an L1 endonuclease-dependent manner. The MHs were longer for 5'-inverted L1 than for 5'-truncated L1, indicating less dependence on annealing in 5'-truncated L1 insertions. CONCLUSIONS: The results suggest that insertions flanked by short TSDs occur more often coupled with the insertion of 5'-truncated L1 than with those of other types of insertions in vivo. The method used in this study can be used to characterize elements without any apparent boundary structures.

Abstract: Chromosome elimination is a process in which some chromatins are discarded from the presumptive somatic cells during early embryogenesis. Eliminated chromatins in hagfish generally consist of repetitive sequences, and they are highly heterochromatinized in germ cells. In this study, we characterized four novel eliminated DNA families, EEPs1-4, from the Taiwanese hagfish Paramyxine sheni. Sequences of these four elements occupied 20-27% of eliminated DNA in total, and each family was arranged mainly in tandem in the germline genome with high copy numbers. Although most of these elements were eliminated, a minor fraction remained in somatic cells. Some eliminated DNA families are shared as eliminated sequences between Eptatretidae and Myxinidae. Fluorescence in situ hybridization (FISH) of these elements showed that not only heterochromatic chromosomes but also both ends of euchromatic chromosomes in germ cells are absent in somatic cells of P. sheni. It strongly suggests that chromosome terminus elimination, in addition to whole chromosome elimination, contributes to somatic chromosome differentiation. Telomere-FISH further showed that chromosome fragmentation and the subsequent de novo addition of telomeric repeats are the likely mechanisms underlying chromosome terminus elimination. These characteristics make it indispensable to study the evolution and mechanisms underlying chromosome elimination in hagfish.

Abstract: Human long interspersed nuclear element-1 (L1) occupies one-sixth of our genome and has contributed to genome evolution in various ways. Approximately 10% of human L1 copies are composed of two L1 segments; the 5' segment and 3' segment are in head-to-head (i.e., 5'-inverted) orientation. Besides mediating their own retrotransposition, L1 has the ability to mobilize mRNA in trans, and the number of retrotransposed mRNA sequences (retrocopies) is estimated to be >6,000. In this study, we identified 48 human-specific retrocopies and 95 chimpanzee-specific retrocopies by comparing the human and chimpanzee genomes. Among these retrocopies, 12 were 5'-inverted. The characteristics of these 5'-inverted retrocopies were similar to those of 5'-inverted L1 copies, indicating that the 5' inversion is generated by the same mechanism. With these findings, we examined the possibility that 5' inversion of the retrocopy generates a new gene that codes for a peptide with a different N terminus. We identified several potential 5'-inverted retrogenes, including those of thymopoietin beta (TMPO) and eukaryotic translation initiation factor 3 subunit 5 (EIF3F). The most interesting candidate was the 5'-inverted retrocopy of small nuclear ribonucleoprotein polypeptide N (SNRPN). This retrocopy was transcribed in the reverse orientation in several organs, had multiple transcript variants, and encoded a protein containing a peptide fragment derived from the N-terminal portion of SNRPN. Our results suggest that mRNA retrotransposition coupled with 5' inversion may be a mechanism to generate new genes distinct from parental genes.

Abstract: Retroelements, elements encoding reverse transcriptase (RT), are ubiquitous in eukaryotes and have a great influence on the evolution of our genome. Detailed information is available on eukaryotic retroelements; however, prokaryotic retroelements are poorly understood. Recently, new types of eukaryotic retroelements were characterized on the basis of their gene composition and their phylogenetic positions. Here we performed a systematic survey to identify novel types of prokaryotic retroelements by analyzing gene neighborhood and protein architecture. We found novel types of gene combination and examined whether they represent actual retroelements. Five monophyletic groups were identified that were distinct from characterized prokaryotic retroelements, showed specific gene combination, were distributed patchily, and included at least 1 example of recent integration. These results strongly indicated the frequent horizontal transfer of these elements. One group encoded DNA polymerase A. A possible function of DNA polymerase A in the life cycle of retroelements is catalyzing second-strand cDNA synthesis, which is DNA polymerization performed using a DNA template not an RNA template. Another group encoded both bacterial primase and carbon-nitrogen hydrolase. Primase is likely to synthesize primers to initiate reverse transcription. Two other groups also encoded carbon-nitrogen hydrolase as a fusion protein with RT. It is difficult to speculate on the function of hydrolase in the life cycle of retroelements. The last group encoded dual RT proteins, which are likely to form heterodimers during replication. The protein sets of these 5 groups of prokaryotic retroelements were completely different from those of eukaryotic retroelements, indicating that the survival constraints of prokaryotic elements were distinct from those of eukaryotic elements. It is likely that these prokaryotic retroelements are maintained as extrachromosomal DNA or RNA or are accidentally integrated into genomes. Our findings presented the possibility that many types of extrachromosomal prokaryotic retroelements remain to be characterized. In addition, we found 8 RT genes were associated with clustered regularly interspaced short palindrome repeats (CRISPRs) of the CRISPR-Cas system. These RT genes are likely to work in immunity against RNA phages via cDNA synthesis.

Abstract: Ribosomal RNA genes are abundant repetitive sequences in most eukaryotes. Ribosomal DNA (rDNA) contains many insertions derived from mobile elements including non-long terminal repeat (non-LTR) retrotransposons. R2 is the well-characterized 28S rDNA-specific non-LTR retrotransposon family that is distributed over at least 4 bilaterian phyla. R2 is a large family sharing the same insertion specificity and classified into 4 clades (R2-A, -B, -C, and -D) based on the N-terminal domain structure and the phylogeny. There is no observation of horizontal transfer of R2; therefore, the origin of R2 dates back to before the split between protostomes and deuterostomes. Here, we in silico identified 1 R2 element from the sea anemone Nematostella vectensis and 2 R2-like retrotransposons from the hydrozoan Hydra magnipapillata. R2 from N. vectensis was inserted into the 28S rDNA like other R2, but the R2-like elements from H. magnipapillata were inserted into the specific sequence in the highly conserved region of the 18S rDNA. We designated the Hydra R2-like elements R8. R8 is inserted at 37 bp upstream from R7, another 18S rDNA-specific retrotransposon family. There is no obvious sequence similarity between targets of R2 and R8, probably because they recognize long DNA sequences. Domain structure and phylogeny indicate that R2 from N. vectensis is the member of the R2-D clade, and R8 from H. magnipapillata belongs to the R2-A clade despite its different sequence specificity. These results suggest that R2 had been generated before the split between cnidarians and bilaterians and that R8 is a retrotransposon family that changed its target from the 28S rDNA to the 18S rDNA.

Abstract: Chromosomal ends of most eukaryotes are composed of simple telomeric repeats. Arthropod telomeres are generally constituted by TTAGG pentanucleotide repeats; however, some insect species including Drosophila melanogaster do not have telomeric repeats. In contrast, the domestic silkworm Bombyx mori contains TTAGG-type telomeric repeats, but the telomerase activity has not been detected in all investigated tissues. To search for a cause of unusual telomere structure in insects, we here identified telomerase reverse transcriptase (TERT) subunit from the domestic silkworm B. mori and the flour beetle Tribolium castaneum. This is the first report of telomerase genes from arthropods. The domestic silkworm TERT gene (BmoTERT) and the flour beetle TERT gene (TcasTERT) both did not have the N-terminal GQ motif. Comparison between cDNA and genomic DNA of BmoTERT revealed that it includes no introns. BmoTERT contains five ATG codons in its 5'UTR, which could reduce the translation of BmoTERT proteins. Also, Northern hybridization indicated that BmoTERT is transcribed at a very low level. These unique features of BmoTERT possibly explain the undetectable Bombyx telomerase activity.

Abstract: Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein. We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.

Abstract: Retrotransposons commonly encode a reverse transcriptase (RT), but other functional domains are variable. The acquisition of new domains is the dominant evolutionary force that brings structural variety to retrotransposons. Non-long-terminal-repeat (non-LTR) retrotransposons are classified into two groups by their structure. Early branched non-LTR retrotransposons encode a restriction-like endonuclease (RLE), and recently branched non-LTR retrotransposons encode an apurinic/apyrimidinic endonuclease-like endonuclease (APE). In this study, we report a novel non-LTR retrotransposon family Dualen, identified from the Chlamydomonas reinhardtii genome. Dualen encodes two endonucleases, RLE and APE, with RT, ribonuclease H, and cysteine protease. Phylogenetic analyses of the RT domains revealed that Dualen is positioned at the midpoint between the early-branched and the recently branched groups. In the APE tree, Dualen was branched earlier than the I group and the Jockey group. The ribonuclease H domains among the Dualen family and other non-LTR retrotransposons are monophyletic. Phylogenies of three domains revealed the monophyly of the Dualen family members. The domain structure and the phylogeny of each domain imply that Dualen is a retrotransposon conserving the domain structure just after the acquisition of APE. From these observations, we discuss the evolution of domain structure of non-LTR retrotransposons.

Abstract: R2 is a non-long-terminal-repeat (LTR) retrotransposon that inserts specifically into 28S rDNA. R2 has been identified in many species of arthropods and three species of chordates. R2 may be even more widely distributed in animals, and its origin may be traceable to early animal evolution. In this study, we identified R2 elements in medaka fish, White Cloud Mountain minnow, Reeves' turtle, hagfish, sea lilies, and some arthropod species, using degenerate polymerase chain reaction methods. We also identified two R2 elements from the public genomic sequence database of the bloodfluke Schistosoma mansoni. One of the two bloodfluke R2 elements has two zinc-finger motifs at the N-terminus; this differs from other known R2 elements, which have one or three zinc-finger motifs. Phylogenetic analysis revealed that the whole phylogeny of R2 can be divided into 11 parts (subclades), in which the local R2 phylogeny and the corresponding host phylogeny are consistent. Divergence-versus-age analysis revealed that there is no reliable evidence for the horizontal transfer of R2 but supports the proposition that R2 has been vertically transferred since before the divergence of the deuterostomes and protostomes. The seeming inconsistency between the R2 phylogeny and the phylogeny of their hosts is due to the existence of paralogous lineages. The number of N-terminal zinc-finger motifs is consistent with the deep phylogeny of R2 and indicates that the common ancestor of R2 had three zinc-finger motifs at the N-terminus. This study revealed the long-term vertical inheritance and the ancient origin of sequence specificity of R2, both of which seem applicable to some other non-LTR retrotransposons.

Abstract: Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)n repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6-8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect.

Abstract: Although most LINEs (long interspersed nuclear elements), which are autonomous non-long-terminal-repeat retrotransposons, are inserted throughout the host genome, three groups of LINEs, the early-branched group, the Tx group, and the R1 clade, are inserted into specific sites within the target sequence. We previously characterized the sequence specificity of the R1 clade elements. In this study, we screened the other two groups of sequence-specific LINEs from public DNA databases, reconstructed elements from fragmented sequences, identified their target sequences, and analyzed them phylogenetically. We characterized 13 elements in the early-branched group and 13 in the Tx group. In the early-branched group, we identified R2 elements from sea squirts and zebrafish in this study, although R2 has not been characterized outside the arthropod group to date. This is the first evidence of cross-phylum distribution of sequence-specific LINEs. The Dong element also occurs across phyla, among arthropods and mollusks. In the Tx group, we characterized five novel sequence-specific families: Kibi for TC repeats, Koshi for TTC repeats, Keno for the U2 snRNA gene, Dewa for the tRNA tandem arrays, and Mutsu for the 5S rRNA gene. Keno and Mutsu insert into the highly conserved region within small RNA genes and destroy the targets. Several copies of Dewa insert different positions of tRNA tandem array, which indicates a certain "site specifier" other than sequence-specific endonuclease. In all three groups, LINEs specific for the rRNA genes or microsatellites can occur as multiple families in one organism. This indicates that the copy number of a target sequence is the primary factor to restrict the variety of sequence specificity of LINEs.

Abstract: Non-long-terminal-repeat (non-LTR) retrotransposons amplify their copies by reverse transcribing mRNA from the 3' end, but the initial processes of reverse transcription are still unclear. We have shown that a telomere-specific non-LTR retrotransposon of the silkworm, SART1, requires the 3' untranslated region (3' UTR) for retrotransposition. With an in vivo retrotransposition assay, we identified several novel motifs within the 3' UTR involved in precise and efficient reverse transcription. Of 461 nucleotides (nt) of the 3' UTR, the central region, from nt 163 to nt 295, was essential for SART1 retrotransposition. Of five putative stem-loops formed in RNA for the SART1 3' UTR, the second stem-loop (nt 159 to 221) is included in this region. Loss of the 3' region (nt 296 to 461) in the 3' UTR and the poly(A) tract resulted in decreased and inaccurate reverse transcription, which starts mostly from several telomeric repeat-like GGUU sequences just downstream of the second stem-loop. These results suggest that short telomeric repeat-like sequences in the 3' UTR anneal to the bottom strand of (TTAGG)(n) repeats. We also demonstrated that the mRNA for green fluorescent protein (GFP) could be retrotransposed into telomeric repeats when the GFP coding region is fused with the SART1 3' UTR and SART1 open reading frame proteins are supplied in trans.

Abstract: Although most non-long terminal repeat (non-LTR) retrotransposons are inserted throughout the host genome, many non-LTR elements in the R1 clade are inserted into specific sites within the target sequence. Four R1 clade families have distinct target specificity: R1 and RT insert into specific sites of 28S rDNA, and TRAS and SART insert into different sites within the (TTAGG)(n) telomeric repeats. To study the evolutionary history of target specificity of R1-clade retrotransposons, we have screened extensively novel representatives of the clade from various insects by in silico and degenerate polymerase chain reaction (PCR) cloning. We found four novel sequence-specific elements; Waldo (WaldoAg1, 2, and WaldoFs1) inserts into ACAY repeats, Mino (MinoAg1) into AC repeats, R6 into another specific site of the 28S rDNA, and R7 into a specific site of the 18S rDNA. In contrast, several elements (HOPE, WISHBm1, HidaAg1, NotoAg1, KagaAg1, Ha1Fs1) lost target sequence specificity, although some of them have preferred target sequences. Phylogenetic trees based on the RT and EN domains of each element showed that (1) three rDNA-specific elements, RT, R6, and R7, diverged from Waldo; (2) the elements having similar target sequences are phylogenetically related; and (3) the target specificity in the R1 clade was obtained once and thereafter altered and lost several times independently. These data indicate that the target specificity in R1 clade retroelements has changed during evolution and is more divergent than has been speculated so far.

Abstract: Telomeres of most insects are composed of simple (TTAGG) (n) repeats that are synthesized by telomerase. However, in some dipteran insects such as Drosophila melanogaster, (TTAGG) (n) repeats or telomerase activity has not been detected. Although telomere structure is well documented in Diptera and Lepidoptera, very limited information is available on lower insect groups. To understand general aspects of telomere function and evolution in insects, we endeavored to characterize structures of the telomeric and subtelomeric regions in a lower insect, the Taiwan cricket, Teleogryllus taiwanemma. FISH analysis of this insect's chromosomes demonstrated (TTAGG) (n) repeat elements in all distal ends. Just proximal to the telomeric repeats, the highly conserved 9-kb long terminal unit (LTU) sequences are tandemly repeated. These were observed in four of six chromosomes, three autosomal ends, and one X-chromosomal end. LTU sequences represent about 0.2% of the T. taiwanemma genome. Each LTU contains a core (TTAGG)(8)-like sequence (TRLS) and five types of conserved sequences-ST (short telomere associated), J (joint), X, SR (satellite sequence rich), and Y-which vary in length from about 150 bp to 2.7 kb. The LTU sequence is defined as ST-J-TRLS-SR-X-Y-X-Y-X. Most LTU regions may be derived from the ancestral common sequence, which is observed in ST regions six times and at many other LTU sites. We could not find the LTU-like sequence in three other crickets including the closest species, T. emma, suggesting that the LTU in T. taiwanemma has been rapidly amplified in subtelomeric regions through recent evolutional events. It is also suggested that the highly conserved structure of the LTU is maintained by recombination and may contribute to telomere elongation, as seen in dipteran insects.