INTRODUCTION

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Telomeres in Drosophila melanogaster are composed of multiple copies of two non-long terminal repeat (LTR) retrotransposons, HeT-A and TART, instead of the short DNA repeats generated by telomerase on the chromosome ends of most eukaryotes (13, 24). Successive transpositions of HeT-A and TART yield arrays of repeats that are larger and more irregular than the repeats produced by telomerase. Nevertheless, these transpositions are, in some sense, equivalent to the telomere-generating action of telomerase; both telomerase and the transposition of HeT-A and TART extend chromosome ends by RNA-templated additions of specific sequences.

HeT-A and TART share two features that distinguish them from other known retrotransposable elements. Both transpose only to the ends of chromosomes (apparently to any chromosome end in D. melanogaster), and each contains a large segment of untranslated sequence (Fig. 1). We have recently shown that, for HeT-A, each of these distinguishing features is also conserved in related species (10), even when phylogenetic separation is great enough for the HeT-A sequences to have diverged by nearly 50%. HeT-A and TART also have some significant differences. TART encodes its own reverse transcriptase (RT); HeT-A does not. However, HeT-A seems efficient in utilizing RT from some other source because most of the documented transpositions onto broken ends have been HeT-A rather than TART (1, 2, 33). A second difference is seen in the large 3' untranslated regions (UTRs). HeT-A elements have a distinctive pattern of A-rich segments in this region (9). Although the sequence of the HeT-A 3' UTR diverges even faster than that of the coding region, the specific pattern of A-rich segments is conserved in other species, suggesting that it has a function (10). The 3' UTRs of TART elements are more heterogeneous; D. melanogaster has at least two subfamilies of TART elements, A and B, whose 3' UTRs do not cross-hybridize (33). TART 3' ends also show much less evidence of sequence patterning than do HeT-A UTRs. However, as discussed below, TART has its own distinctive sequence features.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Diagrams of the two telomeric retrotransposons. The bars under each diagram indicate the sequences used as probes for the RNA hybridization experiments reported here. Each sequence was transcribed in vitro to yield both sense and antisense probes. TART elements can be divided into several families on the basis of their 3' UTR sequences. The two probes marked A and B identify different subfamilies of TART and do not cross-hybridize under the conditions used here. HeT-A elements are ~6 kb, and the TART element shown is >10 kb. (A)n, site of the poly(A) tail on RNA transposition intermediate.

What is now known about the two telomeric retrotransposons raises several questions about function and, ultimately, about evolution. Are the features shared by HeT-A and TART sufficient for either element to fulfill its telomeric function(s)? Are the differences important, so that the two elements must cooperate to form a telomere? Are the two elements derived from a single ancestor, or are they products of convergent evolution? These questions are difficult to answer directly. Every D. melanogaster stock that we have examined has multiple copies of both HeT-A and TART. We detect no pattern in the distribution of the two elements that would suggest either a functional difference or a simple way of breeding a fly with no copies of one of the elements.

To learn more about the relationship between HeT-A and TART, we have extended our studies of the transcription of these elements. We found earlier that the predominant transcript of HeT-A was, as expected for a non-LTR retrotransposon, a sense-strand copy of the entire element (6). We detected no antisense copies of this RNA. Surprisingly, we now find both sense and antisense TART RNAs. Furthermore, the antisense transcripts are more than 10-fold more abundant than sense-strand RNAs. Our analyses indicate that transcriptionally active TART elements are more variable in size and sequence than HeT-A elements; however, the distribution of variants differs among fly stocks, suggesting that these variants are functionally equivalent. The major differences in the sets of transcripts found for HeT-A and TART are not aberrations of the genetic background of a particular stock. We have surveyed several stocks and a distantly related Drosophila species and found the same general patterns.

Another unexpected finding of our studies is an RNA with similarity to the TART RT coding sequence but not to any other part of TART DNA. All the eukaryotic RTs which have been studied, with the notable exception of telomerase, have been encoded by retroelements and transcriptionally linked to the gag coding region. Thus, this new RNA is a novel RT mRNA. HeT-A is an unusual non-LTR retrotransposon in that it does not encode its own RT. It is intriguing that the RT mRNA is found in cells that are expressing HeT-A.

We have found that both subfamilies of TART for which sequence is available have one pair of remarkably conserved repeats. These repeats resemble those found in a small subgroup of non-LTR retrotransposons that have been described as having unequal, or nonidentical, terminal repeats (30). HeT-A does not have repeats of this type. This difference in sequence organization, in conjunction with the differences in transcription patterns, suggests that an ancestral gag sequence has become associated with two different subclasses of non-LTR elements to form HeT-A and TART.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Drosophila stocks and cell lines. Of the D. melanogaster stocks used in this work, 2057 (an isogenic y; cn bw sp stock used for the Drosophila Genome Project P1 phage library [35]) was the generous gift of E. Lozovskaya, and the stocks shown in Fig. 5 were the generous gift of A. A. Hoffmann (20). The other D. melanogaster and Drosophila yakuba stocks have been in our collection for many years. D. melanogaster cultured cells, Schneider line 2, were grown in Dulbecco's modified Eagle's medium with lactalbumin hydrolysate, 10% fetal bovine serum, and 10 mM nonessential amino acids. Schneider line 3 cells were grown in Schneider's medium (Gibco BRL, Gaithersburg, Md.) plus 10% fetal bovine serum.

Sequences for RNA probes (Fig. 1). The following HeT-A probes were from element 23Zn-1 (GenBank accession no. U06920): 5' UTR probe, nucleotides (nt) 982 to 1746; open reading frame (ORF) probe, nt 1746 to 4421; and 3' UTR probe, nt 4851 to 6481. The TART ORF probes were ORF1 (accession no. U14101), nt 1801 to 3336, and ORF2 (accession no. U02279), nt 434 to 2683. The 3' UTR sequence for TART subfamily A was amplified from genomic DNA of stock 2057, and that of subfamily B was amplified from P1 phage 13-14, using PCR primers derived from accession no. U02279 (A probe) and U14101 (B probe). The amplified sequences were cloned in Bluescript II SK (Stratagene, La Jolla, Calif.). For each probe used in this study, sense and antisense strands were transcribed from DNA fragments of identical length.

RNA extraction. One hundred flies were homogenized in a Dounce homogenizer or 2 × 10⁸ to 3 × 10⁸ cultured cells were resuspended in 2 ml of buffer (100 mM NaCl, 50 mM Tris-HCl [pH 7.0], 20 mM EDTA). The solution was brought to 1% sodium dodecyl sulfate (SDS) and 50 µg of Proteinase K/ml. The homogenate was incubated at 37°C for 20 min and then extracted with phenol-chloroform and chloroform. Nucleic acids were precipitated with 3 volumes of ethanol plus 0.2 M LiCl. The pellet was resuspended in 200 µl of H₂O and precipitated with an equal volume of 4 M LiCl at 4°C overnight. Finally, the pellet was resuspended in 200 µL of H₂O and precipitated with 3 volumes of ethanol plus 0.3 M Na acetate.

Northern hybridization. RNA samples (20 µg per lane) were treated with glyoxal, separated on an 0.8% agarose gel, and transferred to Hybond-N nylon membrane according to the method of Sambrook et al. (27). ³²P-labeled riboprobes were transcribed in vitro from DNA fragments inserted into Bluescript II SK with T7 or T3 RNA polymerases, according to the Promega (Madison, Wis.) protocol. Hybridization was performed at 65°C in 4× SET (1× SET is 0.15 M NaCl, 0.03 M Tris-Cl [pH 7.0], and 2 mM EDTA), 5× Denhardt's solution, 0.5% SDS, and 50 µl of salmon sperm DNA/ml (27). The filters were washed three times with 1× SSC (0.15 M NaCl plus 0.015 M sodium citrate) plus 0.5% SDS at 65°C and then treated with 100 U of RNase T₁ (Boehringer Mannheim, Indianapolis, Ind.)/ml in buffer (10 mM Tris-Cl [pH 7.5], 300 mM NaCl, 5 mM EDTA) for 1 h at 37°C, rinsed with 1× SSC-0.5% SDS, and exposed for autoradiography.

Cell fractionation. Schneider 2 cells (2 × 10⁸) were washed once in phosphate-buffered saline, resuspended in 8 ml of homogenization buffer (10 mM Tris HCl [pH 8], 1 mM EDTA, 10 mM NaCl, 10 mM MgCl₂), and placed on ice for 10 min. The cells were disrupted in a Dounce homogenizer and centrifuged for 10 min at 800 × g. The pellet was used as the nuclear fraction. The supernatant was centrifuged again at 800 × g, and the supernatant from this spin was used as the cytoplasmic fraction. RNA was isolated from both the nuclear pellet and the second supernatant, as described above.

Nucleotide sequence accession numbers. The sequences of the clones discussed in this study have been deposited in the GenBank database under accession no. AF072856 (A subfamily, 990-nt probe) and AF072857 (B subfamily, 950-nt probe).

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Technical details of hybridization. It is important to note that both HeT-A and TART are present in the genome in multiple copies with various degrees of divergence. In addition, parts of the noncoding regions of these elements are closely related to sequences in other types of repeats (5). Thus, there are many potential sources for sequences hybridizing with the probes used here, and it is important to eliminate hybridization to similar, but not identical, sequences. For this reason, all hybrids in these experiments were treated with RNase T₁ before autoradiographic exposure. This treatment, adapted from RNase protection experiments, assures that the transcripts detected have strong sequence similarity to the probe, although not necessarily over their complete lengths.

HeT-A elements produce only sense-strand transcripts. When RNA from either flies or cultured cell lines is probed with sequence from any part of HeT-A, the major species of RNA detected is a sense-strand transcript of ~6 kb (Fig. 2). This is the size expected for a full-length transcript of the element. HeT-A elements which have been sequenced differ by multiple insertions and/or deletions (9, 25), but the net result is that the elements are approximately the same size and should be expected to migrate as the slightly broad band seen in our gels (Fig. 2; see also Fig. 7). Two minor bands of apparently larger RNAs detected by these probes are thought to be readthrough transcripts of tandem elements.

View larger version (100K): [in this window] [in a new window]   FIG. 2.   HeT-A elements produce only sense-strand transcripts. Autoradiograph of a Northern blot of total RNA from adult D. melanogaster females (lanes F) and males (lanes M) probed with HeT-A sequences. Single-strand probes detecting sense-strand RNA were used for lanes marked "sense," while "antisense" lanes were probed with the opposite strand. See Fig. 1 for the extent of sequence in each probe. Each probe detects a prominent sense-strand RNA of ~6 kb (arrow). Less abundant RNAs (asterisks) of approximately twice this size and greater may represent readthrough transcripts of tandem elements. (The label at ~2 kb is due to RNA trapped by rRNA.) Each of the probes for sense-strand transcripts also detects one or more small (<2-kb) RNAs. Some of these RNAs appear to be sex specific. No antisense transcripts are detected with any of the probes. The lanes probed for antisense ORF sequences are shown. Similar results were obtained when blots were probed for 5' and 3' UTR antisense sequences. The marker sizes are shown in kilobases.

TART produces both sense and antisense transcripts of a heterogeneous array of full-length elements. In striking contrast to HeT-A, TART produces transcripts of both strands in both flies and cultured cells. We have concentrated our study on the RNAs, both sense and antisense, that run as several distinct bands with estimated sizes between 7.5 and 12 kb (Fig. 3). The numbers, sizes, and relative intensities of these bands differ from stock to stock (compare Fig. 3 through 8). Our studies suggest that all of the RNAs in the 7.5- to 12-kb range represent full-length elements. As discussed below, TART appears to be a more heterogeneous family than HeT-A. TART elements whose 3' UTRs differ by nearly 2 kb in length have been sequenced. The 5' end of TART has not yet been defined. The longest published TART sequence is 10,636 nt (33), and it does not have a complete 5' end. We do not yet have probes for the 5' UTR, but all of the probes that we do have (ORF1, ORF2, and 3' UTR [Fig. 3 and 4]) hybridize to the same bands in this region of the gel; thus, each species of RNA seems to have the entire sequence, even though some are shorter than 10 kb.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   TART produces multiple full-length sequences, both sense and antisense. An autoradiograph is shown of a Northern blot of total RNA from adult Oregon R flies probed for transcripts carrying sequence of TART ORF1 (lanes 1) and ORF2 (lanes 2). Both ORFs are found in the same set of transcripts. Both sense and antisense probes detect sets of transcripts migrating between 7.5 and 12 kb; however, the sense and antisense RNAs do not exactly comigrate. The transcripts in the 7.5- to 12-kb region in Oregon R flies differ (in number and size) from those in other fly stocks and in the two cultured cell lines studied (compare Fig. 3 through 8). Note that the autoradiographic exposures shown are chosen to best display the bands produced by each probe and therefore vary from probe to probe. The detection of sense-strand RNAs requires significantly more exposure than detection of antisense transcripts. The label at ~2 kb is due to RNA trapped by rRNA.

View larger version (119K): [in this window] [in a new window]   FIG. 4.   TART A- and B-subfamily elements yield full-length transcripts of several sizes. An autoradiograph of a Northern blot of total RNA from adult 2057 flies probed with TART sequences is shown. In this stock ORF probes detect two bands of sense-strand RNA migrating above the 9.5-kb marker. (Both ORF1 and ORF2 probes give this result; ORF2 is shown.) The A-subfamily 3' UTR probe identifies the top band as TART A RNA, while the B-subfamily 3' UTR probe labels a band corunning with the lower band but seen only in RNA from males (indicated by the arrow in the B 3' UTR lane). Because females in this stock lack full-length B elements, we conclude that the band just above 9.5 kb hybridizing with ORF probes in females represents a third subfamily of TART found both in males and females. This third-subfamily RNA comigrates with the B transcript in males. Both 3' UTR probes detect very abundant short sense RNAs (<2 kb). Probes for antisense ORF sequences detect two transcripts whose gel mobilities differ slightly from that of the large sense RNAs. The larger antisense transcript may comigrate with the smaller sense RNA. Both of the large antisense RNAs hybridize with A-subfamily 3' UTR probes. The B-subfamily 3' UTR detects a smaller and less abundant antisense RNA seen only in males with either ORF or B 3' UTR probes (indicated by the arrow in the B 3' UTR lane). The label at ~2 kb is due to RNA trapped by rRNA.

TART elements form a heterogeneous family and yield a complex set of transcripts. Both TART (33) and HeT-A (9) can be divided into subfamilies on the basis of their 3' UTR sequences. The intrafamily divergence of TART subfamilies is greater than that of HeT-A subfamilies. The 3' UTRs of different TART subfamilies are unable to cross-hybridize under the conditions used in this work, while different HeT-A subfamily 3' UTRs do cross-hybridize. TART 3' UTRs also have greater length differences than HeT-A elements. The small number of nearly full-length elements which have been sequenced show TART 3' UTRs of 5.1 and 3.3 kb, while HeT-A 3' UTRs are 2.6 to 2.3 kb. These limited data are supported by the sizes of the apparent full-length transcripts of these elements. The TART sense transcripts migrate on gels as though they were 7.5 to 12 kb, while HeT-A transcripts run in a single broad band of ~6.0 kb.

View larger version (90K): [in this window] [in a new window]   FIG. 5.   Elements yielding different-size TART transcripts can be segregated by breeding. An autoradiograph of a Northern blot of total RNA probed for TART antisense ORF2 RNA is shown. Lanes 1 to 6 contain RNA from lines selected from a mass-bred population of D. melanogaster initiated from females collected in Australia in February 1994 (20). Lines 1 to 3 were then selected on the basis of heat shock resistance, and lines 4 to 6 were parallel control lines. (Our samples, a gift of A. Hoffmann, were taken after 3 years of selection.) For our purposes the six lines illustrate the variation in TART expression patterns that can be selected from a large population of flies. Each line shows a somewhat different pattern of TART RNA. Probes for ORF1 show a similar picture. The different patterns seen clearly in the antisense strands are reflected in the sense strands (not shown), although the two strands do not give identical patterns.

TART antisense transcripts are more than 10 times as abundant as sense transcripts. Blots probed for sense-strand RNAs require significantly longer autoradiographic exposures than do blots probed for antisense RNA. To obtain a more quantitative estimate of the relative amounts of the two sets of transcripts, we have used equal amounts of each probe and varied autoradiographic exposures until they yielded bands of nearly equal densities for sense and antisense transcripts. These comparisons show clearly that there is a marked excess of antisense RNAs. Figure 6 shows a typical example of these comparisons. The exposure for the lanes of sense-strand RNAs is six times that for lanes of antisense RNAs. One more factor must be taken into account in estimating the relative amounts of transcripts of the two strands. The sense strands are twice as A rich as the antisense strands. The probes used for these experiments are labeled with [³²P]UTP by in vitro transcription. Therefore, the specific activity of the probe depends on the specific activity of the nucleotide mix and the sequence transcribed. We have used the same nucleotide mix for both sense and antisense probes. The fragment transcribed to give the probe for the sense strand has approximately twice (1.94 times) as many A residues as does its complement, and therefore it will be labeled 1.94 times as heavily as probes for the other strand. Taken together, the probe strength and the exposure time used for Fig. 6 lead to the conclusion that antisense strands are approximately 12 times as abundant as sense strands.

View larger version (68K): [in this window] [in a new window]   FIG. 6.   TART elements produce many more antisense- than sense-strand transcripts. An autoradiograph of a Northern blot of total RNA (replicate preparations) from the D. melanogaster Schneider 2 cell line probed with sequence from TART ORF2 is shown. Autoradiographic exposures were chosen so that probes for sense- and antisense-strand RNA produced bands of approximately equal intensity. The exposure used for sense-strand transcripts (18 h) is approximately six times that for antisense RNA (3 h). Because of the strong strand bias in A residues, the probe used to detect sense-strand RNA has incorporated nearly twice as much 32P as the complementary probe, indicating that antisense transcripts are more than 10-fold more abundant than sense RNA. ORF1 and 3' UTR probes from TART produce similar blots, with the exception that only the ORF2 probe (containing RT sequences) detects a sense-strand transcript of 5.5 kb, which we call RTx (arrow).

TART probes detect an unusual RT mRNA. The RT of retroviruses and retrotransposons is typically encoded by their full-length sense-strand transcripts. The enzyme is translated as part of a polyprotein linked to the product of the gag gene either by a frameshift or by readthrough of a leaky stop codon (14). Full-length TART transcripts partially fit the general pattern. They contain both ORF1, the gag gene, and ORF2, which has the RT coding sequence; however, this ORF2 appears to require an internal initiation for translation, unlike many retrotransposons (33). It was surprising, therefore, to find a sense-strand RNA that hybridized with TART ORF2 probes (Fig. 6) but not with TART sequence either 5' or 3' of the RT sequence (data not shown). Thus, this RNA appears to be an mRNA for RT, and we refer to it as RTx RNA. Because it hybridizes with TART at very high stringency, it might be a processed product. However, if so, it must have undergone removal of both 5' and 3' sequences, something that has not been reported for other retrotransposons. RTx RNA might also be the product of a "free-standing" RT gene, perhaps related to the RT of TART in much the same way that viral oncogenes are related to their cellular counterparts. No antisense copy of RTx RNA has been detected.

The intracellular distribution of TART transcripts differs from that of HeT-A transcripts. Full-length sense-strand transcripts of typical retrotransposons serve as mRNA for the products of the gag and pol genes. The same transcripts can also serve as templates for the reverse transcription required for transposition to a new chromosomal site. For non-LTR retrotransposons, such as HeT-A and TART, this reverse transcription occurs in the nucleus, primed by chromosomal DNA at the site of integration (19). Therefore, the dual roles of full-length transcripts suggest that these transcripts will be found in both the nucleus and the cytoplasm. There is no reason to expect the intracellular distribution of HeT-A transcripts to be different from that of TART transcripts, yet the distributions of these transcripts differ markedly (Fig. 7). When we assay RNA from cell fractions, the sets of presumed full-length TART transcripts, both sense and antisense, are found almost entirely in the nuclear fraction. Therefore, these TART RNAs must be either inside the nucleus or tightly associated with it. In contrast, a significant fraction of full-length HeT-A RNA (which is sense strand only) is present in the cytoplasmic fraction. RTx RNA is also detected in the cytoplasmic fraction, consistent with the supposition that it is an mRNA.

View larger version (99K): [in this window] [in a new window]   FIG. 7.   The nucleocytoplasmic distribution of TART transcripts differs from that of HeT-A transcripts. An autoradiograph of a Northern blot of RNA from cultured cells after nucleocytoplasmic fractionation is shown. N, nuclear fractions; C, cytoplasmic fractions. TART sequences were detected with ORF2 probes. HeT-A sequences were detected with a 3' UTR probe. Full-length HeT-A RNA (right arrow) and TART RTx RNA (left arrow) are found in nuclear and cytoplasmic fractions, while the large TART transcripts (both sense and antisense) are almost entirely limited to the nuclear fraction. The 6-kb HeT-A RNA is seen with all HeT-A probes. The ~4-kb HeT-A RNA is detected only with 3' UTR probes and only in cultured cells; its origin is not known. The label at ~2 kb is due to RNA trapped by rRNA.

Transcription patterns of the telomeric transposons are conserved across species. We have begun our studies of telomeric elements in other species with those of D. yakuba, thought to be separated from D. melanogaster by 5 to 15 million years (15). Previously, we cloned HeT-A elements from D. yakuba (HeT-A^yak) and found that, although the overall sequence identity between HeT-A^yak and HeT-A^mel is only 55%, the D. yakuba HeT-A elements show all the unusual features of HeT-A^mel: they transpose only to telomeres, where they form long arrays; they have the unusual, long 3' UTR; and they lack RT coding sequences (9). Our studies now show that, like HeT-A^mel, HeT-A^yak produces only sense-strand RNA and its full-length transcripts are relatively homogeneous in size, migrating as a single rather broad band in our gels (data not shown).

View larger version (59K): [in this window] [in a new window]   FIG. 8.   The abundant antisense transcription of TART is conserved in related Drosophila species. An autoradiograph of a Northern blot comparing TART transcripts in D. melanogaster (m) with those in D. yakuba (y) is shown. Both samples have been probed with sequences from D. melanogaster TART ORF1 and ORF2. The D. melanogaster lanes were exposed for 20 h, and the D. yakuba lanes were exposed for 44 h. Only lanes with antisense transcripts are shown. In D. yakuba, as in D. melanogaster, TART antisense transcripts are much more abundant than TART sense RNAs.

Comparisons of the HeT-A and TART gag coding sequences give evidence of a common ancestor. The coding region of HeT-A appears to be closely related to ORF1 of TART. Both sequences are clearly related to the ORF1 sequences of several insect non-LTR elements, but HeT-A and TART are most closely related to each other (25). Dot matrix analyses show that the 3' half of the D. melanogaster TART^mel ORF1 has a high level of nucleotide identity with the equivalent part of the coding regions of HeT-A elements from both D. melanogaster and D. yakuba, even though the coding regions of the two HeT-A elements are only ~65% identical to each other at the nucleotide level (Fig. 9).

View larger version (48K): [in this window] [in a new window]   FIG. 9.   TART ORF1 has significant sequence similarity with the HeT-A coding region. (A and B) Dot matrix comparisons of the nucleotide sequence of TART ORF1 from D. melanogaster with sequences of the entire coding regions of HeT-A elements from D. melanogaster (A) and D. yakuba (B). (C) The two HeT-A elements have 65% sequence identity distributed evenly over the coding region. HeT-Amel has 38% identity with TART, and HeT-Ayak has 41% identity with TART. For both HeT-A elements, the regions of identity with TART are strongest in the C-terminal end of the coding region (boxed and marked by stars).

HeT-A and TART belong to different subfamilies of non-LTR retrotransposons. Our sequence analyses reveal that TART displays several features which it does not share with Het-A; instead, these features characterize a small subgroup of non-LTR transposons, elements with unusual terminal repeats (see Fig. 10). A striking feature of non-LTR retrotransposons is their tendency to be truncated at the 5' end. This tendency led to the suggestion that these elements are reverse transcribed into their site of transposition, with variable failure of reverse transcription to complete the copy of the 5' end (31). This basic mechanism of transposition has been elegantly confirmed for the R2Bm element (19). HeT-A and TART are also frequently truncated at the 5' end, although the extent to which this is due to failure of reverse transcription as opposed to erosion of the chromosome end is unclear. The complete 5' end of HeT-A has been defined by the analysis of several elements that had identical junctions with an upstream element, a 5' UTR, and a complete coding region (7).

View larger version (8K): [in this window] [in a new window]   FIG. 10.   Diagrams of TART A and TART B showing locations of perfect repeat sequences (indicated by the black bars under the elements). Both elements are truncated at the 5' end. The TART B diagram is based on data from reference 33;0. (A)n, site of the poly(A) tail on RNA transposition intermediate.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

The Drosophila telomeric transposons present intriguing evolutionary questions. Drosophila telomeres are a striking exception to the almost-universal eukaryotic telomere, which is maintained by the enzyme telomerase. Drosophila must have shared ancestors with organisms that now have telomerase. Therefore, it is interesting to speculate on the evolution of the Drosophila retrotransposon-type telomere. We have suggested that telomere retrotransposons have evolved from components of telomerase (24, 26). This hypothesis has recently received support from evidence that the catalytic subunit of telomerase resembles the RTs of non-LTR retrotransposons (18, 21, 22). This finding is particularly intriguing, since both HeT-A and TART are non-LTR retrotransposons. Nevertheless, there is no compelling evidence to eliminate other possible explanations for the Drosophila telomere, such as the "domestication" of existing, parasitic retrotransposons to replace telomerase in Drosophila or the evolution of telomerase from retrotransposons, with Drosophila demonstrating the ancestral mechanism.

HeT-A and TART belong to different subgroups of non-LTR retrotransposons. The studies reported here offer a new insight into the relationship between HeT-A and TART. TART displays several features which it does not share with HeT-A but which are found in a different subgroup of non-LTR retrotransposons. This evidence that the two elements belong to different subgroups suggests that the evolutions of HeT-A and TART converged by acquiring related gag proteins, which may be responsible for their telomere localization.

Antisense transcripts have been reported for very few non-LTR retrotransposons. From their structure, it would appear that a sense transcript of non-LTR retrotransposons should contain all of the information necessary both for translation of element-specific proteins and for templating of the DNA copy when the element transposes. HeT-A conforms to this expectation. We have studied transcripts in both males and females of several stocks of D. melanogaster, in two immortal cell lines, and in one stock of D. yakuba. In every case we find only sense-strand transcripts of HeT-A. In contrast, parallel studies of TART show, in every case, a large excess of antisense transcripts. Since these studies detect steady-state RNA, the excess could be due to more active promoters for the antisense RNA, to a slower turnover of the RNA, or both.

A novel RNA encoding RT. Our Northern blots show a sense-strand RNA that hybridizes at very high stringency with the probe for TART ORF2 (the RT coding sequence) but does not hybridize with TART sequences either 5' or 3' of the RT gene. Because this RNA appears to encode RT, we have named it RTx RNA. Our data are consistent with three possible explanations for the origin of RTx RNA: (i) it could be transcribed from TART with posttranscriptional processing or altered transcriptional initiation and termination; (ii) it could be the transcript of a new retrotransposon; or (iii) it could be the mRNA product of a cellular gene, as opposed to a retroelement gene. (We refer to this as the "free-standing gene" alternative.)