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Published online before print
September 8, 2006, 10.1101/gr.5348806 Genome Res. 16:1231-1240, 2006 ©2006 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/06 $5.00
Letter Genomic organization of the Drosophila telomere retrotransposable elements1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; 2Berkeley Drosophila Genome Project, Department of Genome Sciences, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
The emerging sequence of the heterochromatic portion of the Drosophila melanogaster genome, with the most recent update of euchromatic sequence, gives the first genome-wide view of the chromosomal distribution of the telomeric retrotransposons, HeT-A, TART, and Tahre. As expected, these elements are entirely excluded from euchromatin, although sequence fragments of HeT-A and TART 3 untranslated regions are found in nontelomeric heterochromatin on the Y chromosome. The proximal ends of HeT-A/TART arrays appear to be a transition zone because only here do other transposable elements mix in the array. The sharp distinction between the distribution of telomeric elements and that of other transposable elements suggests that chromatin structure is important in telomere element localization. Measurements reported here show (1) D. melanogaster telomeres are very long, in the size range reported for inbred mouse strains (averaging 46 kb per chromosome end in Drosophila stock 2057). As in organisms with telomerase, their length varies depending on genotype. There is also slight under-replication in polytene nuclei. (2) Surprisingly, the relationship between the number of HeT-A and TART elements is not stochastic but is strongly correlated across stocks, supporting the idea that the two elements are interdependent. Although currently assembled portions of the HeT-A/TART arrays are from the most-proximal part of long arrays, ~61% of the total HeT-A sequence in these regions consists of intact, potentially active elements with little evidence of sequence decay, making it likely that the content of the telomere arrays turns over more extensively than has been thought.
Telomeres of the genus Drosophila, like those of other eukaryotes, are made up of tandem arrays of nucleotide repeats copied from an RNA template. The distinctive feature of Drosophila telomeres lies in the sequence repeats themselves. In all other studied organisms, telomere repeats are very short simple sequences, which do not code for proteins. In contrast, the Drosophila telomere repeats are two retrotransposable elements, HeT-A and TART (see Pardue and DeBaryshe 2003  ). (In Drosophila melanogaster there are also a few copies of Tahre, an element combining sequences of HeT-A and TART [Abad et al. 2004b]. Tahre has not yet been reported in other Drosophila species.) The Drosophila telomere elements are 2 or 3 orders of magnitude larger than telomere repeats in other organisms, and they encode proteins used for their retrotransposition. HeT-A, TART, and Tahre are non–long terminal repeat (non- LTR) retrotransposons, and the proteins they encode are closely related to the proteins encoded by a group of non-LTR retrotransposons that are abundant in Drosophila genomes. However, HeTA, TART, and Tahre are distinguished from these other retrotransposons in two important ways:
First, HeT-A, TART, and Tahre transpose specifically to chromosome ends, apparently identifying ends by some feature other than DNA sequence. In situ hybridization experiments have not detected these elements in euchromatic regions except when they have bound to the end of a chromosome that has broken in euchromatin (Traverse and Pardue 1988
Second, while most retrotransposons contain very little DNA that does not code for proteins, HeT-A, TART, and Tahre have 3 untranslated regions (3 UTRs) that make up nearly half their sequence. As is typical of UTRs, the sequence of these regions evolves rapidly. Nonetheless, HeT-A and TART arrays maintain the strand composition bias seen on other telomeres—the sense strand is always A+C-rich (Danilevskaya et al. 1998
This combination of unusual, but well-defined, chromosomal distribution and unusual sequence organization suggests that these features are related. The possibility of such a relationship is further emphasized by the fact that fragments of their 3 UTR sequence have been found not only in telomeric heterochromatin but also in other heterochromatic regions of the genome (Danilevskaya et al. 1991
The D. melanogaster euchromatic genome has been completely sequenced (Adams et al. 2000
The Drosophila Heterochromatin Genome Project is extending sequence of the D. melanogaster genome (stock 2057) into heterochromatic regions (Hoskins et al. 2002 The results of this sequence analysis, in conjunction with quantitative hybridization measurements, allowed us to determine the magnitude and range of variation of telomere sequence in flies of different genotypes and in established cell lines. Previous molecular characterization of eukaryotic telomeres produced the unexpected finding that average lengths of telomerase- maintained telomeres fluctuate around equilibrium values specified by the genotype, tissue, and environment. Our measurements of Drosophila telomeres suggest that, despite their unusual retrotransposon mechanism, these telomeres have a similarly dynamic behavior.
HeT-A, TART, and Tahre sequences are not found in euchromatic regions BLAST searches of the finished euchromatic sequence in Release4.2 of the genome confirm in situ hybridization studies showing no HeT-A, TART, or Tahre sequences in euchromatin. There are no sequences with significant similarity (E < 0.002) to HeT-A or Tahre sequences. TART elements have 90 bp in the pol coding region with significant similarity to the pol region of BS, a non- LTR element found in several euchromatic sites. Euchromatin contains a few other sequences with similarity to TART 3 UTRs, but they are so short and scattered that it is likely that they are incidental rather than evidence of TART insertions (see Supplemental material, Section 1). All of these similarities localize to the 3 UTR sequences of TART.
These studies show that HeT-A, TART, and Tahre are strongly excluded from euchromatin. The exclusion of the telomeric elements from a major part of the genome is striking because the euchromatic regions contain elements from at least 76 families of other retrotransposons (Kaminker et al. 2002 In contrast to their presence in euchromatin, other transposable elements are excluded from telomeric HeT-A/TART arrays except for a short transition region at the proximal edge of the array (see below).
Assembled sequence now extends into the HeT-A/TART arrays on the right end of chromosome 4 (4R) and the left endof chromosome X (XL)
Each HeT-A and TART in these two arrays, whether complete or partial, is orientated with its 3 end toward the center of the chromosome. Both the XL and the 4R arrays contain mixtures of complete and partial elements (for details, see Supplemental Table 1). Because partial elements are truncated from the 5 end, ORFs, which all reside near the 5 end, are lost preferentially, and telomere regions are enriched in the 3 UTR sequences that make up an extraordinarily large part of the sequence of these telomere elements (Pardue and DeBaryshe 2003 ). For HeT-A there is also a special class of elements with <62 bp of the 3 -most sequence of the element plus an oligo(A). We call these "tags," and, as discussed below, the unusual HeT-A promoter produces them. Such drastically truncated TART tags are not found, nor are they expected, because TART has a more conventional promoter.
The array on 4R begins only 425 bp distal to the first identified gene (CAPS), whereas the array on XL is separated from the most distal identified gene, Sp71, by a 52.5-kb subtelomeric region containing many transposable elements, including INE-1, Cr1a, and roo but neither HeT-A nor TART. In other D. melanogaster stocks, the sequence immediately interior to HeT-A and TART on the XL telomere is occupied by the repeats of Telomere Associated Sequences (TAS) (Karpen and Spradling 1992 ; Walter et al. 1995). However, in the stock sequenced by the Genome Project (hereafter referred to as 2057) the XL telomere lacks TAS (Abad et al. 2004a). Instead, in this 52.5-kb subtelomeric region, we identify only two small fragments of X TAS (49 bp and 147 bp).
HeT-A and TART elements are almost completely separated from the other sequences in the genome
There is no obvious reason why other non-LTR elements, which are relatively abundant in euchomatin, should not transpose into telomere arrays. In fact, one might suppose that telomere arrays would be safe landing sites because they contain no known vital genes to be disrupted. However, previous studies of HeT-A and TART have not reported these other elements in Drosophila telomeres (see Pardue and DeBaryshe 2003
Much of the assembled sequences consist of complete HeT-A elements
Not unexpectedly, these competent HeT-A elements differ by both nucleotide changes and insertions/deletions (indels). Significant sequence differences are typical of previously cloned HeT-A elements from various (unknown) chromosomal sites (Biessmann et al. 1992
Indels in the 5 and 3 UTRs might not be expected to have much effect on function because these regions are noncoding and have rather repetitive sequences. Surprisingly, there are also a number of indels in the coding sequences, yet none of these cause premature termination of translation. The longest of the encoded proteins is 30 amino acids longer than its shortest homolog, with most of the length difference due to a large indel near the N-terminal end, in a region previously identified as length polymorphic (Pardue et al. 1996
HeT-A tags As expected, eight of the 11 tags in the 4R and XL HeT-A/ TART arrays are immediately 5 of complete elements, either singly or in multiples (Fig. 1; Supplemental Fig. 1). In 4R, three are in tandem before the 5840-bp element (ignoring the 12-bp novel sequence), two are before the 5848-bp element, and one lies directly before the 6012-bp element. In XL a tandem pair precedes the 6006-bp element. We assume these tandem tags originate from multiple transpositions because an element should acquire an additional tag at each transposition. The other three tags are puzzling because none is directly 5 of a complete element; instead they are 5 of truncated HeT-A elements and should have been lost during truncation. We can only speculate on the origin of these last three tags.
A cluster of TARTs on 4R
Surprisingly all the TART A elements in this cluster have >99.9% identical sequences in the regions where they overlap with each other (several are truncated) and with a complete TART A element (AJ566116
[GenBank]
) sequenced by Abad et al. (2004a)
The sequences of these TART A elements provide strong support for the conclusion that the Perfect Non-Terminal Repeats (PNTRs) found at the 5 end and near the 3 end of TART elements are evolving together, somehow achieving the same sequence changes at each end of the element (Danilevskaya et al. 1999
Telomeric fragments in nontelomeric regions of the heterochromatic Y chromosome These nontelomeric HeT-A/TART sequences in the Y heterochromatin differ from those in telomeric arrays in two important ways: First, they are inserted into the chromosome, rather than onto an end. Second, the fragments contain only 3 UTR sequence, yet some elements lack the extreme 3 sequences that appear to be required for transposition. These differences suggest that these nontelomeric HeT-A/TART elements did not transpose into the Y in the way elements transpose to the telomere. n these Y chromosomal regions, HeT-A and TART 3 fragments are associated with other elements (Cr1a, Idefix, Ine-1, X- element, Gypsy, and Stalker), in a way reminiscent of the organization of the transition zone on 4R, suggesting that a segment of such a region could have been moved into the Y in a yet undefined way. HeT-A and TART 3 UTR sequences are a significant fraction of the very small amount of nontelomeric heterochromatic sequence now assembled, whereas essentially none of this sequence is found in euchromatin, striking evidence for our belief that HeT-A and TART sequences cannot enter euchromatin or are detrimental once entered, or both.
Much of the Y chromosome sequence is not yet assembled. We expect more HeT-A and TART fragments to be found because, in addition to the scattered fragments reported here, the Y has at least two large regions of nontelomeric tandem repeats containing HeT-A and TART fragments not yet included in the assembled scaffolds (Danilevskaya et al. 1991
Estimating the number of HeT-A/TART elements in telomere arrays Nevertheless, it is important to have estimates of the total length of telomere transposon sequence and its variation between stocks. We have made such estimates using quantitative Southern hybridization (Fig. 2). All experiments were done entirely with DNA from female flies to avoid sequences on the Y. The blots were probed with HeT-A and TART open reading frame (ORF) sequences, which are the most conserved part of the elements and which also avoid hybridization with 3 UTR sequences in nontelomere regions (for discussion of probe choice, see Methods). DNA was extracted from adult heads because they are a relatively homogeneous source of diploid cells. We obtain similar results from female larval brains (data not shown).
Because the ORF sequences used for probes are near the 5 (distal) end of the element, which is most likely to be truncated, these probes allow an approximate count of full-length elements. More precisely, these hybridization experiments measure the total amount of ORF DNA in the sample (both complete and partial ORFs), which we report in units of full-length ORF ("ORF equivalents"). In conjunction with the fragment distribution measured in the 4R and XL telomere arrays (see Discussion), these data can be used to estimate the number of complete and partial HeT-A or TART elements in the genome. (As discussed later ~80% of the total HeT-A ORF and ~90% of TART ORF is found in complete elements). Tahre is subsumed in the total because of cross- hybridization to HeT-A sequences.
In Figure 3 we show the measured number of HeT-A and TART ORF equivalents in the genome of flies from 2057 (the inbred Canton S stock sequenced by the Genome project), from Oregon R (a commonly used wild-type stock), and from the S2 cell line, derived from Oregon R embryos >30 yr ago (Schneider 1972
The cultured S2 cells have 14 HeT-A and 6 TART elements. Although they have fewer total elements than any of the fly stocks, S2 cells have maintained significant numbers of telomere elements over the >30 yr the line has been in culture. Similar numbers of elements were found in Schneider 3 cells and in a sample of S2 cells that has been frozen for the past six years (data not shown). These results are reminiscent of mammalian immortal cell lines, which tend to have shorter telomere arrays than do primary cultures (Counter et al. 1994 ; Harley et al. 1994; Tommerup et al. 1994).
D. melanogaster telomere length is influenced by genotype
We note that all of the D. melanogaster stocks we have analyzed have both HeT-A and TART elements. TART is always less abundant than HeT-A. An unexpected feature of Figure 4 is the nearly proportional relationship, over a hundredfold range, between the relative amounts of HeT-A and TART across stock lines and tissue type. (Correlation analysis of the nine experiments illustrated in Figure 4 implies that the relative number of HeT-A and TART elements is linearly correlated across genomes at >99% confidence level. [For details, see Supplemental material, Sections 3 and 4.]) Although we believe that HeT-A and TART depend on each other during transposition (Rashkova et al. 2002
Telomere arrays are under-replicated in polytene cells Specifically, we find that salivary gland HeT-A is under- replicated at the 95% confidence level in the Oregon R, 2057, Su(var)2054, and GIII stocks. For TART, pairwise comparison finds under-replication at the 95% confidencelevel for Oregon R and at the 68% confidence level for Su(var)2054, and GIII. For TART in 2057 our data do not show evidence of salivary gland under- replication. Figure 5 shows the replication ratio (the ratio of each element in salivary glands to the same element in heads). When averaged over all stocks HeT-A is significantly less under- replicated than TART. For details, see Supplemental material, Section 3.
HeT-A/TART arrays are easily detectable by in situ hybridization on salivary gland chromosomes, showing that these sequences, like the rest of the chromosome, undergo multiple rounds of replication to make the polytene chromosomes. However, there is reason to believe that new transpositions do not occur in polytene nuclei because HeT-A RNA is not found in these cells (George and Pardue 2003 ), nor does HeT-A Gag enter polytene nuclei (Pardue et al. 2005). Nevertheless, our measurements cannot be explained by a simple failure to replace RNA primers of DNA synthesis (measurements on broken Drosophila chromosome ends average 2 nt per round of replication and are consistent with that mechanism for end shortening) (Levis 1989; Biessmann et al. 1990a). Polytene chromosomes usually undergo a total of  10 rounds of replication and would be almost undetectably shorter after replication at this loss rate Thus, the decreased amount of telomeric DNA we detect in polytenes (Fig.4) must be due to something more than failure to replace these primers.
Intact and apparently functional HeT-A elements are found near the proximal end of the telomere arrays A surprising finding of this study has been the number of apparently functional HeT-A elements deep within the telomere arrays. If addition of telomere repeats serves only to replace eroded sequence on the chromosome end, one would expect sequences deep inside the arrays to decay because, once added to the end, there should be little constraint to maintain function if their only function is to buffer a chromosome end. Instead, the full-length sequences here have maintained ORFs and other regions needed for function. The existence of functional elements in proximal regions of these long telomere arrays suggests that these interior sequences may be renewed more frequently than has been thought and that turnover in these arrays does not simply replace terminal sequence lost in DNA replication. A likely possibility is that telomeres sometimes undergo drastic shortening, perhaps by a mechanism such as that proposed for yeast telomere rapid deletion (Lustig 2003 ) or mammalian t-loop homologous recombination (Wang et al. 2004). This shortening would then be followed by rapid multiple transpositions to restore the telomere to an appropriate length. Such a drastic shortening might also explain the loss of most of the TASs on the 2057 XL telomere.
More HeT-A and TART elements than expected have intact 5 ends
The data do not support the expectation that significantly more telomere elements would be truncated; 70% (14 of 20) of our HeT-A and 71% (5 of 7) of our TART elements are truncated, slightly less than the 79% seen for elements not subject to end erosion. For this calculation, we omit the tiny "tags," which we believe are byproducts of the unusual HeT-A promoter. These tags all have <50 bp of homology and therefore would also have been omitted in the calculation of Kaminker et al. (2002)
This observation that a significant fraction of HeT-A elements in the array shows little, if any, terminal erosion suggests that ends are protected from degradation or that transpositions frequently occur in rapid succession before erosional loss. These possibilities are not mutually exclusive. Protection could be provided by terminal structures like the t-loops seen on chromosomes in other organisms (Griffith et al. 1999
D. melanogaster has very long telomere arrays
That estimate has several uncertainties. Apparently intact elements can differ by indels that add up to several hundred base pairs; the 5 end of TART presents technical problems because of its PNTRs (see Pardue and DeBaryshe 2003
Using these numbers, we calculate from our hybridization results that the 2057 genome contains polarized HeT-A/TART arrays with ~29 complete HeT-A elements and approximately seven complete TART elements. Correcting for partial elements, we calculate ~365 kb of total HeT-A and TART sequence on eight telomeres, an average of ~45.6 kb of HeT-A and TART sequence per telomere. Perforce, we use the same correction factors for estimates of other genomes.
We can compare this estimate to the one other measurement done for D. melanogaster telomere arrays. Abad et al. (2004a)
Although most eukaryotes have very similar telomere sequences, multicellular eukaryotes have much longer telomere arrays than do unicellular eukaryotes. Among the longest studied telomeres are those of inbred strains of laboratory mice. These telomeres range from 30–150 kb (Kipling and Cooke 1990 It is interesting that mice and flies, the two organisms known to have unusually long telomeres, are also unusual because they have been kept in small isolated laboratory populations for many years, suggesting that something about the population structure or relatively luxurious laboratory conditions may affect telomere length. It will be interesting to see whether wild- derived D. melanogaster have shorter telomeres, like wild-derived mice.
D. melanogaster telomere length is influenced by genotype
The retrotransposon telomeres of Drosophila, similar to those maintained by telomerase, have genetically modulated length control. Savitsky et al. (2002)
Tel-1 mutant flies have significantly more telomeric DNA than the other stocks, and the amounts are influenced by genetic background, as seen by comparing the Gaiano and GIII strains (Gaiano is the source of the Tel-1 mutation, which was moved into Oregon R to make GIII) (Siriaco et al. (2002)
Analysis of the assembled sequence suggested that Drosophila telomeres occasionally undergo large deletions of the type reported in yeast and humans (Lustig 2003
Fly stocks and cell lines Oregon R, a wild-type stock maintained in our laboratory for many years; 2057, an isogenic y1 , cn1 , bw, 1sp1 Canton S stock obtained from A. Villasante (Centro Servero Ochoa); GIII, Tel-1 mutant in Oregon R background made by J. Mason (NIEHS) (Siriaco et al. 2002 ); Su(var)2054/CyO, y+ , obtained from J. Eissenberg (St. Louis University). The Schneider S2 and S3 cell lines (Schneider 1972) have been in our laboratory for >30 and 3 yr, respectively.
Sequence analyses The telomeric sequence assemblies (4th and X) have been validated by comparison to restriction fingerprint digests (BACN05O16, CH221–48I20, respectively) using five enzymes: ApaLI, BamHI, EcoRI, HindIII, and XhoI (collaboration between the Berkeley Drosophila Genome Project and BC Cancer Agency Genome Sciences Center and BC Cancer Agency, Vancouver, Canada [in prep.]).
Probes for Southern hybridization These probes allow direct comparison of genomes on the basis of their content of ORF coding sequence. Information from the sequence assembly can then be used to derive an estimation of complete and partial elements from these ORF equivalents.
Southern hybridization analyses Hybridization was in 4 SET (1 SET is 0.15 M NaCl, 2 mM EDTA, 0.03 M Tris-Cl at pH 7.0), 2 Denhart's solution, 0.5% SDS, and 100 µg salmon sperm DNA per milliliter at 65°C. Washes were 1 SSC (0.15 M NaCl, 0.015 M Na citrate at pH 7.0) and 0.5% SDS at 65°C. Hybridized 32P-labeled probes were detected by scanning the filters with Molecular Dynamics PhosphorImagers, and the data were reduced to usable digital form by Molecular Dynamics ImageQuant software. In reducing the data, background subtraction, utilizing measuremen t of interlane exposure adjacent to each lane of DNA, was necessary, primarily to avoid overestimation of the rp49-probed DNA measurements and, hence, underestimation of the quantity of HeT-A and TART DNA present. Gels used to determine the "gold standard" absolute number of ORF equivalents per genome (in S2 cells and Oregon R and 2057 Female Adult Heads) also contained three lanes each of the rp49 and retrotransposon probes, which were used for determining the relative activity of each probe. Actually, as discussed above and in detail in Supplemental material, Section 4, we report the number of ORF equivalents per genome in each stock,i.e., the total length of ORF sequence, both full length and partial, measured in units of full-length ORF sequence. For this set of measurements, two independent experiments were performed. This procedure gave multiple independent measurements of each stock for each probe. Data processing was performed using an Excel spreadsheet developed for the purpose. For other samples, three lanes of Oregon R Female Adult Heads were run on each gel as a control, and the number of genome equivalents per stock was determined relative to OregonR. The actual number of ORF equivalents was then determined by multiplying by the reference value determined for Oregon R heads in the more extensive measurements described above. Data from the six probe lanes were also analyzed to demonstrate that our experimental techniques (filter preparation, digitizing, and analysis) are linear over the full range of DNA deposited in the gel. Measurements were linear within a few percent over all source strengths (measured in counts per pixel) (data not shown). For details of the data analysis and statistical controls used, see Supplemental material, Section 4.
We thank J. Eissenberg for the Su(var)2054 stock, J. Mason for the GIII stock, and A. Villasante for the 2057 stock. We are grateful to R.A. Hoskins, C.D. Smith, J. Carlson, and A.B. de Carvalho for helpful advice. We thank E. Casacuberta, J. Collett, R. Dudley, and K. Lowenhaupt for stimulating discussions. This work has been supported by National Institutes of Health Grant GM50315 to M.L.P. The sequencing portion of this work was supported by NIH grant P50-HG00750 (G.M.R.) and carried out under U.S. Department of Energy contract DE-AC0376SF00098.
3 Corresponding author.
E-mail mlpardue{at}mit.edu; fax (617) 253-8699. Supplemental material is available online at http://www.genome.org. Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.5348806.
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ABSTRACT
Results
The average length of the complete HeT-A element is 5893 169 bp (SD) (based on the seven elements listed in Supplemental Table 2). 
88%) and lacks the very large polymorphic region in ORF1. We have searched the genome sequence with the exact sequences of the two probes used, and the only detectable possible cross reacting sequence is 69 nt in ORF2 of the BS element, which has 76% identity with the TART probe. BS is a minor element; there are eight copies of this sequence in the sequenced genome so even if it cross-reacts, it contributes little to the total. 
- und 
-Heterochromatin sowie Konstanz und Bau der Chromomeren bei Drosophila . Biol. Zent. Bl. 54: 588–609.