TAHRE, a Novel Telomeric Retrotransposon from Drosophila melanogaster, Reveals the Origin of Drosophila Telomeres
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分子生物学进展 2004年第9期
* Centro de Biología Molecular Severo Ochoa (CBMSO), CSIC-UAM, Madrid, Spain
Servicio Interdepartamental de Investigación (SIdI), UAM, Madrid, Spain
Children's Hospital Oakland Research Institute (CHORI) Oakland, CA, USA.
E-mail: avillasante@cbm.uam.es
Abstract
Drosophila telomeres do not have typical telomerase repeats. Instead, two families of non-LTR retrotransposons, HeT-A and TART, maintain telomere length by occasional transposition to the chromosome ends. Despite the work on Drosophila telomeres, its evolutionary origin remains controversial. Herein we describe a novel telomere-specific retroelement that we name TAHRE (Telomere-Associated and HeT-A-Related Element). The structure of the three telomere-specific elements indicates a common ancestor. These results suggest that preexisting transposable elements were recruited to perform the cellular function of telomere maintenance. A recruitment similar to that of a retrotransposal reverse transcriptase has been suggested as the common origin of telomerases.
Key Words: telomeres ? telomeric retrotransposons ? Drosophila
Introduction
Drosophila telomeres lack the telomerase-generated repeats characteristic of other eukaryotic chromosomes. Instead, telomere length is maintained by the occasional transposition to the chromosome tips of the non-LTR retrotransposons HeT-A and TART (Mason and Biessmann 1995; Pardue et al. 1996). These two retrotransposons are the only transposable elements known to have a vital cellular function. HeT-A and TART transpose only to chromosome ends, probably by target-primed reverse transcription (Biessmann et al. 1992b; Levis et al. 1993). The targeting does not seem to depend on the DNA sequence at the transposition site (Traverse and Pardue 1988; Biessmann et al. 1990; Biessmann et al. 1992a; Sheen and Levis 1994). TART has the two open reading frames (ORFs) typical of many non-LTR retrotransposons: ORF1 and ORF2 (also called gag and pol because of sequence similarities to retroviral gag and pol genes). The ORF2 has both endonuclease and reverse transcriptase (RT) domains. However, HeT-A is an atypical element because it has no ORF2, so the RT for its transposition is produced in trans from an unknown source. As a distinctive feature, HeT-A and TART have an unusually long 3' untranslated region (UTR) that in the case of the HeT-A contains imperfect repeats. The transcription of HeT-A seems to require a promoter located in the 3' UTR of another upstream element (Danilevskaya et al. 1997). Although the HeT-A elements are transcribed in the normal 5' to 3' direction, TART elements show sense and anti-sense transcription from promoters still unidentified (Danilevskaya et al. 1999). The function of these anti-sense transcripts is unknown. HeT-A is several times more abundant than TART, and they appear to be randomly mixed in the head-to-tail arrays originated by successive transposition. Fragments of these telomeric elements are also found in centric heterochromatin (Traverse and Pardue 1989; Danilevskaya et al. 1993; Levis et al. 1993; Losada et al. 1997; Agudo et al. 1999; Losada et al. 1999), but never in euchromatin.
The recent identification of TART and HeT-A elements in Drosophila virilis has shown that these elements have been performing the cellular function of telomere maintenance for more than 60 million years (Casacuberta and Pardue 2003a, b). Nevertheless, the evolutionary origin of these telomeric retrotransposons remains controversial. Thus, although the ORF1 of HeT-A and TART are closely related (Danilevskaya et al. 1999), it has been suggested that the common features of these elements result from convergent evolution and not from evolution from a common ancestor (Danilevskaya et al. 1999; Pardue and DeBaryshe 2002; Casacuberta and Pardue 2003b). In addition, it has been hypothesized that HeT-A derives from telomerase encoding sequences, in distinction to the alternative mechanism that HeT-A derived from preexisting retrotransposons by losing its RT (Pardue et al. 1996; Pardue and DeBaryshe 1999; 2002; 2003).
In this report we describe a novel retroelement that implies a common origin for known telomeric retrotransposons.
Materials and Methods
Strain and DNA Analysis
In this work, two sheared-DNA BAC libraries from the Drosophila melanogaster isogenic strain yellow (y1); cinnabar (cn1) brown (bw1) speck (sp1) were used: CHORI221 (http://bacpac.chori.org/droso221.htm) and CHORI223 (http://bacpac.chori.org/drososmall223.htm). The libraries were screened with use of colony hybridization by the Church and Gilbert method (Church and Gilbert 1984) with a probe isolated by polymerase chain reaction (PCR) from the distal end of BACR40C07 (library RCPI-98) (primers pair 5'-GGAGGTCATATATTAAAGGG-3' and 5'-TAATACGACTCACTATAGGG-3'). This probe contains nucleotides 4840-6295 from GenBank entry AJ54281. Membranes containing the arrays of clones from each library were provided by the Children's Hospital Oakland Research Institute BACPAC resources. The probe was 32P-labeled by random-priming and the hybridization performed overnight at 68oC. For restriction enzyme analysis of BACs, both conventional 0.8% agarose gel electrophoresis and pulse–field gel electrophoresis (PFGE) were used. PFGE was performed in a CHEF-DRII apparatus (Biorad, Hercules, CA) on 1% agarose gels in 0.5X TBE (1xTBE is 90 mM Tris-borate/2 mM EDTA) for 20 h using 150 V and a 14 s pulse time.
DNA Sequencing and Sequence Analysis
The sequencing strategy was primer walking on BAC DNA and PCR products (for primers see supplementary table 1). The sequence from the distal end of BACR40C07 allowed us to design primers to initiate the sequencing of the ORF2 of the new element. Consequently, using BACs from different locations (containing only one copy of TAHRE), we were able to sequence the ORF2 and part of its 5' and 3' flanking sequences. To extend these sequences, PCR fragments were obtained from the appropriated BACs. For the full-length copy of TAHRE, three PCR products were generated: a 3.3-kb fragment from the 3' UTR of HeT-A to a region of the ORF1 specific of TAHRE; a 4.3-kb fragment from the ORF2 of TAHRE to the 3' UTR of HeT-A; and a 2.5-kb fragment from the 3' UTR of TAHRE to the ORF1 of HeT-A. The ORF1, ORF2, and part of the 3' UTR of the truncated copies of TAHRE were sequenced directly from BACs and the rest of the 3' UTR from the corresponding PCR fragments. All sequencing was performed using big dye-termination reagents and ABI/PE 377 automated sequencers.
Sequences were analyzed by Blast searches. Alignments were performed with ClustalX (Thompson et al. 1997), followed by minor manual adjustments of gaps. Phylogenetic trees were generated by the Neighbor-Joining (NJ) method by using MEGA 2.1 (Kumar et al. 2001). Dot-matrix analysis was performed with the COMPARE and DOTPLOT programs from the CGC package.
Results and Discussion
The isolation and characterization of a large number of BACs from the distal-most chromosome regions of the telomeres of the y; cn bw sp strain of D. melanogaster has allowed us to complete the assembly at the telomeres (Abad et al. unpublished data). In the course of our searches of the D. melanogaster database with the BAC end sequences of all isolated clones, we found that one particular clone from the XL telomere, BACR40C07, carries an end sequence with 65% nucleotide identity to the RT domain of TART elements. This sequence might either derive from a nontelomeric element inserted in the XL telomere, or correspond to a novel TART-related retrotransposon. To distinguish between these alternatives, we screened two sheared-DNA BAC libraries with a PCR probe from this end sequence. This screening identified multiple telomeric clones from the XL, 2L, and 2R telomeres (fig. 1) and two overlapping clones of unknown location (CHORI223-07A04 and CHORI223-43O02). By using Southern blotting we identified four elements in telomeres (two in XL, one in 2L, and one in 2R; see fig. 1) and two adjacent elements in the unknown location. Partial sequencing of CHORI223-07A04 and CHORI223-43O02 showed that they contain two degenerated elements carrying stop codons. Of the four telomeric elements, only one is complete (XL1); the other three are truncated by varying amounts at their 5' ends (fig. 2). In all cases there is a 3' end of an HeT-A element upstream of these elements. Therefore, they clearly represent a novel telomeric retrotransposon. The coding regions in each of these elements are open and differ between elements by several mutations, which in some cases change the amino acid sequence. The elements contain patterns of mutations and indels that suggest an evolutionary relationship wherein the one at 2L would be the oldest.
FIG. 1. Location of a novel telomeric retrotransposon at the telomeres of the y; cn bw sp isogenic strain. Telomeric contigs were joined to release 3.1 of the Drosophila Genome Project by fingerprinting and partial sequence analysis (Abad et al. unpublished data). Only BACs containing the novel telomeric element (TAHRE) are shown. Short vertical lines indicate sites for EcoRI. Large vertical lines marked by N indicate NotI sites. The restriction fragments labeled in black are positive in Southern hybridization with the probe from the distal end of BACR40C07. The different telomeric elements are shown in colors: red for HeT-A, yellow for TART, and blue for TAHRE. Individual TAHRE elements are labeled according to their localizations from the tip of the chromosome. TAS = telomeric-associated sequences
FIG. 2. Structure of the four TAHRE elements found at the telomeres of the y; cn bw sp isogenic strain. XL1 and XL2 indicate the first and second TAHRE elements from the chromosome tip of the XL telomere. 2R and 2L indicate the TAHRE elements from 2R and 2L telomeres, respectively. The nucleotide differences and the indels between the sequences of the element copies are shown as short vertical lines and as larger vertical lines, respectively. Distances (bp) from the 5' end of the XL1 TAHRE element are indicated below the element. The cluster of CCHC boxes in ORF1 and the endonuclease and RT domains in ORF2 are also indicated
Database comparisons show that although the ORF2 of the novel element is similar to that of TART, the 5' UTR, ORF1, and 3' UTR are very similar to the corresponding sequences of HeT-A, (fig. 3A). For this reason we named this new element TAHRE (Telomere-Associated and HeT-A-Related Element). The 5' UTR of TAHRE has just one of the AT-rich regions found in the 5' UTR of HeT-A elements; the ORF1 does not require large gaps to optimize alignment with the ORFs of HeT-A subfamilies, and the 3' UTR has a larger number of imperfect repeats than any described HeT-A element (fig. 3A). TAHRE shares with TART the presence of a short spacer between the stop codon of ORF1 and the initiation codon of ORF2, suggesting that the translation of TAHRE ORF2 may require an internal ribosomal initiation as has been suggested for TART (Sheen and Levis 1994).
FIG. 3. Comparative analysis of TAHRE sequences against other retrotransposons. (A) Dot matrix comparisons showing the nucleotide similarity between TAHRE, HeT-A, and a full-length copy of TART A. (B) Phylogenetic relationships of Gag protein sequences from TAHRE, HeT-A subfamilies, and D. yakuba HeT-A. (C) Phylogenetic relationships of RT domains, including TART elements from different Drosophila species, TAHRE, jockey, F, G, I, R1, R2, 1731, copia, and gypsy-like retrotransposons
To analyze the phylogenetic relationships between the Gag proteins of TAHRE and HeT-As, their complete coding regions (for alignments see supplementary fig. 1) were compared by using NJ algorithms (Kumar et al. 2001). The Gag protein of TAHRE is more similar to that of the HeT-A subfamilies (Biessmann et al. 1994; Pardue et al. 1996; Abad et al. unpublished data) from D. melanogaster than to the Gag protein of the HeT-A from Drosophila yakuba (Danilevskaya et al. 1998) (fig. 3B). On the other hand, by using seven previously defined RT domains (Xiong and Eickbush 1990) (for alignments see supplementary fig. 2), we have constructed a phylogenetic tree that includes TAHRE and other 19 retroelements. This analysis identifies TAHRE as a member of the jockey clade but divergent from TART (fig. 3C). The similarity of the ORFs, ORF1 and ORF2, of TART and TAHRE indicates that they had a common ancestor. Moreover, HeT-A may have derived from TAHRE by a deletion event or by retrotransposition of a spliced subgenomic RNA coding for ORF1. We favor the second scenario because retroelements are known to generate spliced RNAs coding for Gag- or Env-like proteins that occasionally could serve as transposition intermediates (Yoshioka et al. 1991).
Drosophila telomeric retrotransposons share telomere-specific transposition and large 3' UTRs, but they seem to have evolved to fulfill specialized tasks (fig. 4). Once telomerase has been lost and the recombination-based mechanism to maintain chromosome termini was acting normally (Cohn and Edstrom 1992; Kahn et al. 2000), telomere erosion could activate the mobilization of retroelements via a DNA-damage signaling pathway (Rudin and Thompson 2001; Scholes et al. 2003), that will eventually restore telomere function by its addition to the ends (Yamamoto et al. 2003). This ancestral telomeric element has evolved to optimize telomere maintenance. We propose that a processed copy of TAHRE without RT (proto-HeT-A) has evolved to become the main source of RNA templates; the unique complete copy of TAHRE could supply the RT that controls HeT-A retrotransposition, and it is possible that the anti-sense transcripts from TART may function in controlling double-stranded RNA-mediated telomere heterochromatinization (Aravin et al. 2001). Although these elements have diverged to perform specific missions, they are functioning in concert. Interestingly, in this strain we have found that the unique full-length copy of TAHRE, a full-length copy of HeT-A, and the longest copy of TART A (Abad et al. unpublished data) are all together in a cluster at the X telomere (pink circle in figs. 1 and 4). The future analysis of other strains will help to understand the significance of this telomeric retrotransposon complex.
FIG. 4. Possible evolution of Drosophila melanogaster telomeric retrotransposons from an ancestral non-LTR retroelement
In conclusion, the structure of TAHRE suggests how existing non-LTR retrotransposons could have been recruited to perform the cellular function of telomere maintenance. In this process, the retrotransposal RT has become co-opted to prevent loss of telomeric sequences. A similar recruitment of the RT of non-LTR retrotransposons has been suggested as the common origin of telomerases (Zimmerly et al. 1995; Eickbush 1997). Finally, the functional diversification of the Drosophila telomeric retrotransposons through evolution could be considered as a recent example of an ancestral mechanism that led to telomerase.
Acknowledgements
We thank R. Levis, D. Gubb, C. H. Langley, P. Ripoll, and I. Marin for critical comments and suggestions. This work was supported by grant BMC2002-02632 from the Dirección General de Investigación del MCYT (A.V.), a grant from the Fundación Ramón Areces (A.M.-G.), and an institutional grant from the Fundación Ramón Areces.
The GenBank accession numbers for the sequences reported in this paper are AJ542581-4, AJ549733-4, AJ549753-4, and AJ564682-7.
Literature Cited
Agudo, M., A. Losada, J.P. Abad, S. Pimpinelli, P. Ripoll, and A. Villasante. 1999. Centromeres from telomeres? The centromeric region of the Y chromosome of Drosophila melanogaster contains a tandem array of telomeric HeT-A- and TART-related sequences. Nucl. Acids Res. 27:3318-3324.
Aravin, A.A., N.M. Naumova, A.V. Tulin, V.V. Vagin, Y.M. Rozovsky, and V.A. Gvozdev. 2001. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster genome. Curr. Biol. 11:1017-1027.
Biessmann, H., L.E. Champion, M. O'Hair, K. Ikenaga, B. Kasravi, and J.M. Mason. 1992a. Frequent transpositions of Drosophila melanogaster HeT-A transposable elements to receding chromosome ends. EMBO J. 11:4459-4469.
Biessmann, H., B. Kasravi, T. Bui, G. Fugiwara, L. E. Champion, and J. M. Mason. 1994. Comparison of two active HeT-A retroposons of Drosophila melanogaster. Chromosoma 103:90-98.
Biessmann, H., K. Valgeirsdottir, A. Lofsky, C. Chin, B. Ginther, R.W. Levis, and M.-L. Pardue. 1992b. HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster. Mol. Cell. Biol. 12:3910-3918.
Biessmann, H., J.M. Mason, K. Ferry, M. d'Hulst, K. Valgeirsdottir, K.L. Traverse, and M.-L. Pardue. 1990. Addition of telomere-associated HeT DNA sequences "heals" broken chromosome ends in Drosophila. Cell 61:663-673.
Casacuberta, E., and M.-L. Pardue. 2003a. Transposon telomeres are widely distributed in the Drosophila genus: TART elements in the virilis group. Proc. Natl. Acad. Sci. USA 100:3363-3368.
Casacuberta, E., and M.-L. Pardue. 2003b. HeT-A elements in Drosophila virilis: retrotransposon telomeres are conserved across the Drosophila genus. Proc. Natl. Acad. Sci. USA 100:14091-14096.
Church, G.M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995.
Cohn, M., and J.-L. Edstrom. 1992. Telomere-associated repeats in Chironomus form discrete subfamilies generated by gene conversion. J. Mol. Evol. 35:114-132.
Danilevskaya, O.N., I.R. Arkhipova, K.L. Traverse, and M.-L. Pardue. 1997. Promoting in tandem: the promoter for telomere transposon HeT-A and implications for the evolution of retroviral LTRs. Cell 88:647-655.
Danilevskaya, O.N., T. Chen, J. Wong, M. Alibhai, and M.-L. Pardue. 1998. Unusual features of the Drosophila melanogaster telomere transposable element HeT-A are conserved in Drosophila yakuba telomere elements. Proc. Natl. Acad. Sci. USA 95:3770-3775.
Danilevskaya, O., A. Lofsky, E.V. Kurenova, and M.-L. Pardue. 1993. The Y chromosome of Drosophila melanogaster contains a distinctive subclass of Het-A-related repeats. Genetics 134:531-543.
Danilevskaya, O.N., K.L. Traverse, N.C. Hogan, P.G. DeBaryshe, and M.-L. Pardue. 1999. The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol. Cell. Biol. 19:873-881.
Eickbush, T.H. 1997. Telomerase and retrotransposons: which came first? Science 277:911-912.
Kahn, T., M. Savitsky, and P. Georgiev. 2000. Attachment of HeT-A sequences to chromosomal termini in Drosophila melanogaster may occur by different mechanisms. Mol. Cell. Biol. 20:7634-7642.
Kumar, S., K. Tamura, I.B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.
Levis, R.W., R. Ganesan, K. Houtchens, L.A. Tolar, and F.-M. Sheen. 1993. Transposons in place of telomeric repeats at a Drosophila telomere. Cell 75:1083-1093.
Losada, A., J.P. Abad, and A. Villasante. 1997. Organization of DNA sequences near the centromere of the Drosophila melanogaster Y chromosome. Chromosoma 106:503-512.
Losada, A., M. Agudo, J.P. Abad, and A. Villasante. 1999. HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3. Mol. Gen. Genet. 262:618-622.
Mason, J.M., and H. Biessmann. 1995. The unusual telomeres of Drosophila. Trends Genet. 11:58-62.
Pardue, M.-L., O.N. Danilevskaya, K. Lowenhaupt, F. Slot, and K.L. Traverse. 1996. Drosophila telomeres: new views on chromosome evolution. Trends Genet. 12:48-52.
Pardue, M.-L., and P.G. DeBaryshe. 1999. Telomeres and telomerase: more than the end of the line. Chromosoma 108:73-82.
Pardue, M.-L., and P.G. DeBaryshe. 2002. Telomeres and transposable elements. Pp. 870-887 in N.L. Craig, ed. Mobile DNA II. ASM Press, Washington, D.C.
Pardue, M.-L., and P.G. DeBaryshe. 2003. Retrotransposons provide an evolutionary robust non-telomerase mechanism to maintain telomeres. Annu. Rev. Genet. 37:485-511.
Rudin, C.M., and C.B. Thompson. 2001. Transcriptional activation of short interspersed elements by DNA-damaging agents. Genes, Chromosomes Cancer 30:64-71.
Scholes, D.T., A.E. Kenny, E.R. Gamache, Z. Mou, and M.J. Curcio. 2003. Activation of an LTR-retrotransposon by telomere erosion. Proc. Natl. Acad. Sci. USA 100:15736–15741.
Sheen, F.-M., and R.W. Levis. 1994. Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini. Proc. Natl. Acad. Sci. USA 91:12510-12514.
Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and D.G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 25:4876-4882.
Traverse, K.L., and M.-L. Pardue. 1988. A spontaneously opened ring chromosome of Drosophila melanogaster has acquired He-T DNA sequences at both new telomeres. Proc. Natl. Acad. Sci. USA 85:8116-8120.
Traverse, K.L., and M.-L. Pardue. 1989. Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes. Chromosoma 97:261-271.
Xiong, Y., and T.H. Eickbush. 1990. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9:3353-3362.
Yamamoto, Y., Y. Fujimoto, R. Arai, M. Fujie, S. Usami, and T. Yamada. 2003. Retrotransposon-mediated restoration of Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes. Nucl. Acids Res. 31:4646-4653.
Yoshioka, K., H. Kanda, H. Akiba, M. Enoki, and T. Shiba. 1991. Identification of an unusual structure in the Drosophila melanogaster transposable element copia: evidence for copia transposition through an RNA intermediate. Gene 103:179-184.
Zimmerly, S., H. Guo, P.S. Perlman, and A.M. Lambowitz. 1995. Group II introns mobility occurs by target DNA-primed reverse transcription. Cell 82:545-554.(José P. Abad*,1, Beatriz )
Servicio Interdepartamental de Investigación (SIdI), UAM, Madrid, Spain
Children's Hospital Oakland Research Institute (CHORI) Oakland, CA, USA.
E-mail: avillasante@cbm.uam.es
Abstract
Drosophila telomeres do not have typical telomerase repeats. Instead, two families of non-LTR retrotransposons, HeT-A and TART, maintain telomere length by occasional transposition to the chromosome ends. Despite the work on Drosophila telomeres, its evolutionary origin remains controversial. Herein we describe a novel telomere-specific retroelement that we name TAHRE (Telomere-Associated and HeT-A-Related Element). The structure of the three telomere-specific elements indicates a common ancestor. These results suggest that preexisting transposable elements were recruited to perform the cellular function of telomere maintenance. A recruitment similar to that of a retrotransposal reverse transcriptase has been suggested as the common origin of telomerases.
Key Words: telomeres ? telomeric retrotransposons ? Drosophila
Introduction
Drosophila telomeres lack the telomerase-generated repeats characteristic of other eukaryotic chromosomes. Instead, telomere length is maintained by the occasional transposition to the chromosome tips of the non-LTR retrotransposons HeT-A and TART (Mason and Biessmann 1995; Pardue et al. 1996). These two retrotransposons are the only transposable elements known to have a vital cellular function. HeT-A and TART transpose only to chromosome ends, probably by target-primed reverse transcription (Biessmann et al. 1992b; Levis et al. 1993). The targeting does not seem to depend on the DNA sequence at the transposition site (Traverse and Pardue 1988; Biessmann et al. 1990; Biessmann et al. 1992a; Sheen and Levis 1994). TART has the two open reading frames (ORFs) typical of many non-LTR retrotransposons: ORF1 and ORF2 (also called gag and pol because of sequence similarities to retroviral gag and pol genes). The ORF2 has both endonuclease and reverse transcriptase (RT) domains. However, HeT-A is an atypical element because it has no ORF2, so the RT for its transposition is produced in trans from an unknown source. As a distinctive feature, HeT-A and TART have an unusually long 3' untranslated region (UTR) that in the case of the HeT-A contains imperfect repeats. The transcription of HeT-A seems to require a promoter located in the 3' UTR of another upstream element (Danilevskaya et al. 1997). Although the HeT-A elements are transcribed in the normal 5' to 3' direction, TART elements show sense and anti-sense transcription from promoters still unidentified (Danilevskaya et al. 1999). The function of these anti-sense transcripts is unknown. HeT-A is several times more abundant than TART, and they appear to be randomly mixed in the head-to-tail arrays originated by successive transposition. Fragments of these telomeric elements are also found in centric heterochromatin (Traverse and Pardue 1989; Danilevskaya et al. 1993; Levis et al. 1993; Losada et al. 1997; Agudo et al. 1999; Losada et al. 1999), but never in euchromatin.
The recent identification of TART and HeT-A elements in Drosophila virilis has shown that these elements have been performing the cellular function of telomere maintenance for more than 60 million years (Casacuberta and Pardue 2003a, b). Nevertheless, the evolutionary origin of these telomeric retrotransposons remains controversial. Thus, although the ORF1 of HeT-A and TART are closely related (Danilevskaya et al. 1999), it has been suggested that the common features of these elements result from convergent evolution and not from evolution from a common ancestor (Danilevskaya et al. 1999; Pardue and DeBaryshe 2002; Casacuberta and Pardue 2003b). In addition, it has been hypothesized that HeT-A derives from telomerase encoding sequences, in distinction to the alternative mechanism that HeT-A derived from preexisting retrotransposons by losing its RT (Pardue et al. 1996; Pardue and DeBaryshe 1999; 2002; 2003).
In this report we describe a novel retroelement that implies a common origin for known telomeric retrotransposons.
Materials and Methods
Strain and DNA Analysis
In this work, two sheared-DNA BAC libraries from the Drosophila melanogaster isogenic strain yellow (y1); cinnabar (cn1) brown (bw1) speck (sp1) were used: CHORI221 (http://bacpac.chori.org/droso221.htm) and CHORI223 (http://bacpac.chori.org/drososmall223.htm). The libraries were screened with use of colony hybridization by the Church and Gilbert method (Church and Gilbert 1984) with a probe isolated by polymerase chain reaction (PCR) from the distal end of BACR40C07 (library RCPI-98) (primers pair 5'-GGAGGTCATATATTAAAGGG-3' and 5'-TAATACGACTCACTATAGGG-3'). This probe contains nucleotides 4840-6295 from GenBank entry AJ54281. Membranes containing the arrays of clones from each library were provided by the Children's Hospital Oakland Research Institute BACPAC resources. The probe was 32P-labeled by random-priming and the hybridization performed overnight at 68oC. For restriction enzyme analysis of BACs, both conventional 0.8% agarose gel electrophoresis and pulse–field gel electrophoresis (PFGE) were used. PFGE was performed in a CHEF-DRII apparatus (Biorad, Hercules, CA) on 1% agarose gels in 0.5X TBE (1xTBE is 90 mM Tris-borate/2 mM EDTA) for 20 h using 150 V and a 14 s pulse time.
DNA Sequencing and Sequence Analysis
The sequencing strategy was primer walking on BAC DNA and PCR products (for primers see supplementary table 1). The sequence from the distal end of BACR40C07 allowed us to design primers to initiate the sequencing of the ORF2 of the new element. Consequently, using BACs from different locations (containing only one copy of TAHRE), we were able to sequence the ORF2 and part of its 5' and 3' flanking sequences. To extend these sequences, PCR fragments were obtained from the appropriated BACs. For the full-length copy of TAHRE, three PCR products were generated: a 3.3-kb fragment from the 3' UTR of HeT-A to a region of the ORF1 specific of TAHRE; a 4.3-kb fragment from the ORF2 of TAHRE to the 3' UTR of HeT-A; and a 2.5-kb fragment from the 3' UTR of TAHRE to the ORF1 of HeT-A. The ORF1, ORF2, and part of the 3' UTR of the truncated copies of TAHRE were sequenced directly from BACs and the rest of the 3' UTR from the corresponding PCR fragments. All sequencing was performed using big dye-termination reagents and ABI/PE 377 automated sequencers.
Sequences were analyzed by Blast searches. Alignments were performed with ClustalX (Thompson et al. 1997), followed by minor manual adjustments of gaps. Phylogenetic trees were generated by the Neighbor-Joining (NJ) method by using MEGA 2.1 (Kumar et al. 2001). Dot-matrix analysis was performed with the COMPARE and DOTPLOT programs from the CGC package.
Results and Discussion
The isolation and characterization of a large number of BACs from the distal-most chromosome regions of the telomeres of the y; cn bw sp strain of D. melanogaster has allowed us to complete the assembly at the telomeres (Abad et al. unpublished data). In the course of our searches of the D. melanogaster database with the BAC end sequences of all isolated clones, we found that one particular clone from the XL telomere, BACR40C07, carries an end sequence with 65% nucleotide identity to the RT domain of TART elements. This sequence might either derive from a nontelomeric element inserted in the XL telomere, or correspond to a novel TART-related retrotransposon. To distinguish between these alternatives, we screened two sheared-DNA BAC libraries with a PCR probe from this end sequence. This screening identified multiple telomeric clones from the XL, 2L, and 2R telomeres (fig. 1) and two overlapping clones of unknown location (CHORI223-07A04 and CHORI223-43O02). By using Southern blotting we identified four elements in telomeres (two in XL, one in 2L, and one in 2R; see fig. 1) and two adjacent elements in the unknown location. Partial sequencing of CHORI223-07A04 and CHORI223-43O02 showed that they contain two degenerated elements carrying stop codons. Of the four telomeric elements, only one is complete (XL1); the other three are truncated by varying amounts at their 5' ends (fig. 2). In all cases there is a 3' end of an HeT-A element upstream of these elements. Therefore, they clearly represent a novel telomeric retrotransposon. The coding regions in each of these elements are open and differ between elements by several mutations, which in some cases change the amino acid sequence. The elements contain patterns of mutations and indels that suggest an evolutionary relationship wherein the one at 2L would be the oldest.
FIG. 1. Location of a novel telomeric retrotransposon at the telomeres of the y; cn bw sp isogenic strain. Telomeric contigs were joined to release 3.1 of the Drosophila Genome Project by fingerprinting and partial sequence analysis (Abad et al. unpublished data). Only BACs containing the novel telomeric element (TAHRE) are shown. Short vertical lines indicate sites for EcoRI. Large vertical lines marked by N indicate NotI sites. The restriction fragments labeled in black are positive in Southern hybridization with the probe from the distal end of BACR40C07. The different telomeric elements are shown in colors: red for HeT-A, yellow for TART, and blue for TAHRE. Individual TAHRE elements are labeled according to their localizations from the tip of the chromosome. TAS = telomeric-associated sequences
FIG. 2. Structure of the four TAHRE elements found at the telomeres of the y; cn bw sp isogenic strain. XL1 and XL2 indicate the first and second TAHRE elements from the chromosome tip of the XL telomere. 2R and 2L indicate the TAHRE elements from 2R and 2L telomeres, respectively. The nucleotide differences and the indels between the sequences of the element copies are shown as short vertical lines and as larger vertical lines, respectively. Distances (bp) from the 5' end of the XL1 TAHRE element are indicated below the element. The cluster of CCHC boxes in ORF1 and the endonuclease and RT domains in ORF2 are also indicated
Database comparisons show that although the ORF2 of the novel element is similar to that of TART, the 5' UTR, ORF1, and 3' UTR are very similar to the corresponding sequences of HeT-A, (fig. 3A). For this reason we named this new element TAHRE (Telomere-Associated and HeT-A-Related Element). The 5' UTR of TAHRE has just one of the AT-rich regions found in the 5' UTR of HeT-A elements; the ORF1 does not require large gaps to optimize alignment with the ORFs of HeT-A subfamilies, and the 3' UTR has a larger number of imperfect repeats than any described HeT-A element (fig. 3A). TAHRE shares with TART the presence of a short spacer between the stop codon of ORF1 and the initiation codon of ORF2, suggesting that the translation of TAHRE ORF2 may require an internal ribosomal initiation as has been suggested for TART (Sheen and Levis 1994).
FIG. 3. Comparative analysis of TAHRE sequences against other retrotransposons. (A) Dot matrix comparisons showing the nucleotide similarity between TAHRE, HeT-A, and a full-length copy of TART A. (B) Phylogenetic relationships of Gag protein sequences from TAHRE, HeT-A subfamilies, and D. yakuba HeT-A. (C) Phylogenetic relationships of RT domains, including TART elements from different Drosophila species, TAHRE, jockey, F, G, I, R1, R2, 1731, copia, and gypsy-like retrotransposons
To analyze the phylogenetic relationships between the Gag proteins of TAHRE and HeT-As, their complete coding regions (for alignments see supplementary fig. 1) were compared by using NJ algorithms (Kumar et al. 2001). The Gag protein of TAHRE is more similar to that of the HeT-A subfamilies (Biessmann et al. 1994; Pardue et al. 1996; Abad et al. unpublished data) from D. melanogaster than to the Gag protein of the HeT-A from Drosophila yakuba (Danilevskaya et al. 1998) (fig. 3B). On the other hand, by using seven previously defined RT domains (Xiong and Eickbush 1990) (for alignments see supplementary fig. 2), we have constructed a phylogenetic tree that includes TAHRE and other 19 retroelements. This analysis identifies TAHRE as a member of the jockey clade but divergent from TART (fig. 3C). The similarity of the ORFs, ORF1 and ORF2, of TART and TAHRE indicates that they had a common ancestor. Moreover, HeT-A may have derived from TAHRE by a deletion event or by retrotransposition of a spliced subgenomic RNA coding for ORF1. We favor the second scenario because retroelements are known to generate spliced RNAs coding for Gag- or Env-like proteins that occasionally could serve as transposition intermediates (Yoshioka et al. 1991).
Drosophila telomeric retrotransposons share telomere-specific transposition and large 3' UTRs, but they seem to have evolved to fulfill specialized tasks (fig. 4). Once telomerase has been lost and the recombination-based mechanism to maintain chromosome termini was acting normally (Cohn and Edstrom 1992; Kahn et al. 2000), telomere erosion could activate the mobilization of retroelements via a DNA-damage signaling pathway (Rudin and Thompson 2001; Scholes et al. 2003), that will eventually restore telomere function by its addition to the ends (Yamamoto et al. 2003). This ancestral telomeric element has evolved to optimize telomere maintenance. We propose that a processed copy of TAHRE without RT (proto-HeT-A) has evolved to become the main source of RNA templates; the unique complete copy of TAHRE could supply the RT that controls HeT-A retrotransposition, and it is possible that the anti-sense transcripts from TART may function in controlling double-stranded RNA-mediated telomere heterochromatinization (Aravin et al. 2001). Although these elements have diverged to perform specific missions, they are functioning in concert. Interestingly, in this strain we have found that the unique full-length copy of TAHRE, a full-length copy of HeT-A, and the longest copy of TART A (Abad et al. unpublished data) are all together in a cluster at the X telomere (pink circle in figs. 1 and 4). The future analysis of other strains will help to understand the significance of this telomeric retrotransposon complex.
FIG. 4. Possible evolution of Drosophila melanogaster telomeric retrotransposons from an ancestral non-LTR retroelement
In conclusion, the structure of TAHRE suggests how existing non-LTR retrotransposons could have been recruited to perform the cellular function of telomere maintenance. In this process, the retrotransposal RT has become co-opted to prevent loss of telomeric sequences. A similar recruitment of the RT of non-LTR retrotransposons has been suggested as the common origin of telomerases (Zimmerly et al. 1995; Eickbush 1997). Finally, the functional diversification of the Drosophila telomeric retrotransposons through evolution could be considered as a recent example of an ancestral mechanism that led to telomerase.
Acknowledgements
We thank R. Levis, D. Gubb, C. H. Langley, P. Ripoll, and I. Marin for critical comments and suggestions. This work was supported by grant BMC2002-02632 from the Dirección General de Investigación del MCYT (A.V.), a grant from the Fundación Ramón Areces (A.M.-G.), and an institutional grant from the Fundación Ramón Areces.
The GenBank accession numbers for the sequences reported in this paper are AJ542581-4, AJ549733-4, AJ549753-4, and AJ564682-7.
Literature Cited
Agudo, M., A. Losada, J.P. Abad, S. Pimpinelli, P. Ripoll, and A. Villasante. 1999. Centromeres from telomeres? The centromeric region of the Y chromosome of Drosophila melanogaster contains a tandem array of telomeric HeT-A- and TART-related sequences. Nucl. Acids Res. 27:3318-3324.
Aravin, A.A., N.M. Naumova, A.V. Tulin, V.V. Vagin, Y.M. Rozovsky, and V.A. Gvozdev. 2001. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster genome. Curr. Biol. 11:1017-1027.
Biessmann, H., L.E. Champion, M. O'Hair, K. Ikenaga, B. Kasravi, and J.M. Mason. 1992a. Frequent transpositions of Drosophila melanogaster HeT-A transposable elements to receding chromosome ends. EMBO J. 11:4459-4469.
Biessmann, H., B. Kasravi, T. Bui, G. Fugiwara, L. E. Champion, and J. M. Mason. 1994. Comparison of two active HeT-A retroposons of Drosophila melanogaster. Chromosoma 103:90-98.
Biessmann, H., K. Valgeirsdottir, A. Lofsky, C. Chin, B. Ginther, R.W. Levis, and M.-L. Pardue. 1992b. HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster. Mol. Cell. Biol. 12:3910-3918.
Biessmann, H., J.M. Mason, K. Ferry, M. d'Hulst, K. Valgeirsdottir, K.L. Traverse, and M.-L. Pardue. 1990. Addition of telomere-associated HeT DNA sequences "heals" broken chromosome ends in Drosophila. Cell 61:663-673.
Casacuberta, E., and M.-L. Pardue. 2003a. Transposon telomeres are widely distributed in the Drosophila genus: TART elements in the virilis group. Proc. Natl. Acad. Sci. USA 100:3363-3368.
Casacuberta, E., and M.-L. Pardue. 2003b. HeT-A elements in Drosophila virilis: retrotransposon telomeres are conserved across the Drosophila genus. Proc. Natl. Acad. Sci. USA 100:14091-14096.
Church, G.M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995.
Cohn, M., and J.-L. Edstrom. 1992. Telomere-associated repeats in Chironomus form discrete subfamilies generated by gene conversion. J. Mol. Evol. 35:114-132.
Danilevskaya, O.N., I.R. Arkhipova, K.L. Traverse, and M.-L. Pardue. 1997. Promoting in tandem: the promoter for telomere transposon HeT-A and implications for the evolution of retroviral LTRs. Cell 88:647-655.
Danilevskaya, O.N., T. Chen, J. Wong, M. Alibhai, and M.-L. Pardue. 1998. Unusual features of the Drosophila melanogaster telomere transposable element HeT-A are conserved in Drosophila yakuba telomere elements. Proc. Natl. Acad. Sci. USA 95:3770-3775.
Danilevskaya, O., A. Lofsky, E.V. Kurenova, and M.-L. Pardue. 1993. The Y chromosome of Drosophila melanogaster contains a distinctive subclass of Het-A-related repeats. Genetics 134:531-543.
Danilevskaya, O.N., K.L. Traverse, N.C. Hogan, P.G. DeBaryshe, and M.-L. Pardue. 1999. The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol. Cell. Biol. 19:873-881.
Eickbush, T.H. 1997. Telomerase and retrotransposons: which came first? Science 277:911-912.
Kahn, T., M. Savitsky, and P. Georgiev. 2000. Attachment of HeT-A sequences to chromosomal termini in Drosophila melanogaster may occur by different mechanisms. Mol. Cell. Biol. 20:7634-7642.
Kumar, S., K. Tamura, I.B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.
Levis, R.W., R. Ganesan, K. Houtchens, L.A. Tolar, and F.-M. Sheen. 1993. Transposons in place of telomeric repeats at a Drosophila telomere. Cell 75:1083-1093.
Losada, A., J.P. Abad, and A. Villasante. 1997. Organization of DNA sequences near the centromere of the Drosophila melanogaster Y chromosome. Chromosoma 106:503-512.
Losada, A., M. Agudo, J.P. Abad, and A. Villasante. 1999. HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3. Mol. Gen. Genet. 262:618-622.
Mason, J.M., and H. Biessmann. 1995. The unusual telomeres of Drosophila. Trends Genet. 11:58-62.
Pardue, M.-L., O.N. Danilevskaya, K. Lowenhaupt, F. Slot, and K.L. Traverse. 1996. Drosophila telomeres: new views on chromosome evolution. Trends Genet. 12:48-52.
Pardue, M.-L., and P.G. DeBaryshe. 1999. Telomeres and telomerase: more than the end of the line. Chromosoma 108:73-82.
Pardue, M.-L., and P.G. DeBaryshe. 2002. Telomeres and transposable elements. Pp. 870-887 in N.L. Craig, ed. Mobile DNA II. ASM Press, Washington, D.C.
Pardue, M.-L., and P.G. DeBaryshe. 2003. Retrotransposons provide an evolutionary robust non-telomerase mechanism to maintain telomeres. Annu. Rev. Genet. 37:485-511.
Rudin, C.M., and C.B. Thompson. 2001. Transcriptional activation of short interspersed elements by DNA-damaging agents. Genes, Chromosomes Cancer 30:64-71.
Scholes, D.T., A.E. Kenny, E.R. Gamache, Z. Mou, and M.J. Curcio. 2003. Activation of an LTR-retrotransposon by telomere erosion. Proc. Natl. Acad. Sci. USA 100:15736–15741.
Sheen, F.-M., and R.W. Levis. 1994. Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini. Proc. Natl. Acad. Sci. USA 91:12510-12514.
Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and D.G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 25:4876-4882.
Traverse, K.L., and M.-L. Pardue. 1988. A spontaneously opened ring chromosome of Drosophila melanogaster has acquired He-T DNA sequences at both new telomeres. Proc. Natl. Acad. Sci. USA 85:8116-8120.
Traverse, K.L., and M.-L. Pardue. 1989. Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes. Chromosoma 97:261-271.
Xiong, Y., and T.H. Eickbush. 1990. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9:3353-3362.
Yamamoto, Y., Y. Fujimoto, R. Arai, M. Fujie, S. Usami, and T. Yamada. 2003. Retrotransposon-mediated restoration of Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes. Nucl. Acids Res. 31:4646-4653.
Yoshioka, K., H. Kanda, H. Akiba, M. Enoki, and T. Shiba. 1991. Identification of an unusual structure in the Drosophila melanogaster transposable element copia: evidence for copia transposition through an RNA intermediate. Gene 103:179-184.
Zimmerly, S., H. Guo, P.S. Perlman, and A.M. Lambowitz. 1995. Group II introns mobility occurs by target DNA-primed reverse transcription. Cell 82:545-554.(José P. Abad*,1, Beatriz )