| Nucleic Acids Research | Pages |
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
Introduction
Materials And Methods
Drosophila strains
DNA analysis, sequencing and probes
Fluorescence in situ hybridization (FISH) to metaphase chromosomes
Results
Identification of a YAC clone derived from the centromeric region of the Y using the FISH technique
The YAC yw6F11 contains 3.1 kb tandem repeat units made of HeT-A and TART 3[prime]-non-coding sequences
Organization of the heterochromatin near the 18HT repeat cluster
The region h18 appears to be conserved in the Y chromosome of D.simulans
Discussion
Acknowledgements
References
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
Received May 4, 1999; Revised and Accepted June 24, 1999
DDBJ/EMBL/GenBank accession nos. AJ009983, AJ009984
ABSTRACT Cytological and cytogenetic studies have previously defined the region needed for centromeric function in the Y chromosome of Drosophila melanogaster. We have identified a YAC clone that originated from this region. Molecular analysis of the YAC and genomic DNAs has allowed the description of a satellite DNA made of telomeric HeT-A- and TART-derived sequences and the construction of a long-range physical map of the heterochromatic region h18. Sequences within the YAC clone are conserved in the centromeric region of the sibling species Drosophila simulans. That telomere-derived DNA now forms part of the centromeric region of the Y chromosome could indicate a telomeric origin of this centromere. The existence of common determinants for the function of both centromeres and telomeres is discussed.
INTRODUCTION
Over 67 years ago, cytological observations on the formation of ring chromosomes by McClintock (1) suggested that the centromere was a repetitive structure. The molecular evidence for this conclusion has just emerged through the structural analysis of rearranged centromeres from humans (2,3), maize (4) and Drosophila (5,6). Transfection studies with synthetic arrays of [alpha] satellite DNA (7) and with YACs containing [alpha] satellite DNA from the human 21 centromere region (8) suggested that repeated sequences alone were sufficient to form the centromere of multicellular eukaryotes. However, a very careful molecular analysis of a human neocentromere has shown that no satellite DNA is present in it (9), indicating that [alpha] satellite is not necessary for human centromere function and implying that additional epigenetic factors are, at least in some abnormal instances, sufficient. An involvement of epigenetic mechanisms in centromeric function has also been deduced from studies in Schizosaccharomyces pombe and Drosophila melanogaster: a partial centromere from S.pombe can switch between an active and an inactive state (10) and acentric mini-chromosomes that exhibit neocentromere activity have been found in D.melanogaster (11). Thus, the presence of rare neocentromeres in humans and Drosophila points to a dynamic nature of the DNA sequences required for centromeric function. This fact, together with the global change undergone by any satellite DNA via inevitable expansions-contractions (12), may explain the failure in finding discernible homology between centromeric DNA sequences throughout evolution. Drosophila melanogaster has been taken as a paradigm of centromeric diversity since each centromeric region seems to carry different repeated sequences (6,13). However, that centromere-specific satellites are not present in all centromeres might hold for D.melanogaster but not for other Drosophila species: the centromeric dodeca satellite of D.melanogaster is present in this species only in the centromeric region of chromosome 3 (14), but homologous sequences are found at both sides of the centromeric regions of chromosomes 2 and 3 in the sibling species Drosophila simulans (15). If, as it is generally admitted, metacentric chromosomes arose from Robertsonian fusion of ancestor acrocentric chromosomes, one ancestor of D.simulans had to have four acrocentric chromosomes carrying dodeca satellite-related sequences in all of its centromeres, a situation similar to that which we nowadays see in the autosomal centromeres of Drosophila hydei (16).
Centromeric regions in vertebrates (17) and plants (18,19) have been shown to contain telomeric-like sequences. These results could suggest a centromeric role for these sequences or be simply a reflection of Robertsonian fusions; however, in no case has it been technically possible to assay a centromeric function of these telomeric-like sequences nor has it been proposed that Robertsonian fusions could be a way to generate centromeric activity by modification of previous telomeric functions. Telomeric sequences have also been described in internal regions of the Y chromosome of D.melanogaster, even though these sequences are quite different from the ones found in the large majority of species (20,21). In most eukaryotic chromosomes, telomeres are composed of variable numbers of simple repeat sequences characterized by clusters of G residues on the 3[prime]-end of each strand of chromosomal DNA. These sequences are synthesized by a cellular reverse transcriptase called telomerase (22); this ensures that chromosomes can compensate for the incomplete DNA replication of their ends and conserve intact their neighboring coding regions. Drosophilids and other dipterans have slightly different, but in the long run equivalent, mechanisms to conserve subtelomeric integrity by adding non-coding sequences to chromosome ends (23). In the case of D.melanogaster, the telomeres are made primarily of sequences of the non-LTR retrotransposon HeT-A (HeT-A elements lack an ORF responsible for their own retrotransposition; 24,25) added to the tip of all chromosomes through still unknown molecular mechanisms after reverse transcription of RNAs derived from the so-called `master genes' (26). Besides HeT-A elements, some telomeres also carry sequences of TART, another non-LTR telomeric-specific retrotransposon (27). In this report we will show that a tandem repeat of HeT-A- and TART-related sequences is present in the centromeric region of the Y.
MATERIALS AND METHODS
Drosophila strains
Oregon R was used as the wild-type strain. The other stock used in this work was XYS.YL, Df(1)259, y- w/ C(1)RA l(1)J1 In(1)sc8/ Dp(1;f)1187, y+. For mutations and rearrangements see Lindsley and Zimm (28). T(Y;2)A77p is a derivative of T(Y;2)A77 caused by spontaneous deletion of regions h1-h16 in the YP2D element of the translocation (29).
DNA analysis, sequencing and probes
High molecular weight DNA from 0-12 h Drosophila embryos, diploid tissues of male and female third instar larvae, and yeast chromosomes were prepared in agarose plugs as described (14,30). The DNA restriction enzyme analysis was performed by pulsed-field gel electrophoresis (PFGE) or by conventional gel electrophoresis as previously described (31). Signal quantification of autoradiograms were carried out by volume integration of digitized images using a phosphorimager. The YAC library employed in this study has been described by Abad et al. (14). The polymerase chain reaction (PCR) technique was used to obtain sequences from the YAC yw6F11. PCR reactions were carried out as described by Losada et al. (31). The oligonucleotide primers used in the amplification reactions were: HeT-A box, 5[prime]-CTGTCTCCGTACCTCCACCAGCAAAGTTAA-3[prime]; HeT-A box rev, 5[prime]-AGTAAATTCTGTTCCGCATCCAC-3[prime]; TART-2, 5[prime]-GCTTGCTGCCTACTGGTATCTC-3[prime]; TART-4, 5[prime]-ATAAGACTATAGTTGATGGCGG-3[prime].
Primer 1091 from the YAC vector arm has already been described (31). The fragments obtained by PCR were cloned in vector pGEM-T and sequenced using the T7 polymerase kit. Sequence analysis was performed using the University of Wisconsin GCG package version 8.0. The probe from the Circe element was pGcirce8 (31). The probe for the transposable element Copia was a gift from S. Campuzano. The probe from the F element was a gift from M. Berloco. The HeT-A element probes, 2b and ORF I+II, were contributed by O. Danilevskaya (25). The TART probe was a gift from R. Levis (32). The dodeca satellite probe was pBK6E218 (14).
Fluorescence in situ hybridization (FISH) to metaphase chromosomes
Neuroblast chromosomes were prepared as previously described (33). YAC probes used for FISH were gel-purified and labeled by random priming with biotin-16-UTP or digoxigenin-11-UTP as described (14). Hybridization and detection were done following protocols previously described (33). Biotinylated probes and digoxigenin-labeled probes were detected with FluoroLink CY3-avidin and anti-digoxigenin-FITC, respectively. Chromosomes were counterstained with 4[prime],6-diamino-2-phenylindole (DAPI). Slides were analyzed using a Zeiss Axioplan epifluorescence microscope equipped with a cooled CCD camera. The fluorescent signals from the CY3, FITC and DAPI staining were recorded separately as gray scale digital images and then pseudocolored and merged using the Adobe Photoshop program.
RESULTS
Identification of a YAC clone derived from the centromeric region of the Y using the FISH technique
In a study of prometaphase mitotic chromosomes stained with different dyes Gatti and Pimpinelli (34) subdivided the heterochromatic Y of D.melanogaster into 25 regions (h1-h25) as schematically shown in Figure 1C. Using Y-autosome translocations they were able to locate the centromeric function in the interval h17-h18. When stained with Hoescht 3358 or DAPI, regions h17-h18 usually appear as a single brightly fluorescent block; the reasons for subdividing this block into two regions were the occasional presence of a small (not a primary) constriction in the middle of the block after staining with Hoescht 3358 and the duller staining of h18 with quinacrine. Previous studies have shown the presence of decayed HeT-A retrotransposons at the junction of regions h18 and h19 (31) and the presence of TART-related sequences in region h16 (S.Pimpinelli, unpublished results). These sequences flank the centromeric region of the Y and thus provide entry points to search for even more centric sequences. In order to isolate these more centromeric sequences we screened our Drosophila YAC library (14) for HeT-A- and TART-containing clones. The colony hybridizations were performed consecutively with DNA fragments from the two telomeric retrotransposons: the HeT-A probe was 2b, a 1.3 kb fragment between nucleotides 6109 and 7422 from the 3[prime]-UTR of a HeT-A element (accession no. U06947), and the TART probe was a 2.2 kb SacI fragment between nucleotides 434 and 2683 from the ORF2 of a TART element (U02279). A total of 10 clones cross-hybridized strongly with HeT-A sequences, five clones with TART sequences and only one was positive for both probes.
Figure 1. Cytogenetic localization of clone yw6F11 in D.melanogaster. (A and B) In situ hybridization of the YAC to prometaphase chromosomes from Oregon R males and T(Y;2)A77p, respectively. Hybridization signals are shown in red on the chromosomes counterstained with DAPI (blue). Inserts in (A) and (B) show another example of the chromosome labeled by the probe and its corresponding DAPI stain to facilitate the localization of the signal. (C and D) Localization of the YAC on the cytogenetic maps of the Y and the YP2D element of T(Y;2)A77p, respectively. The diagrams represent the fluorescence of DAPI-stained Y chromosomes. Black blocks are brightly fluorescing, hatched regions dully fluorescing and empty regions are non-fluorescing. The position of the centromere (CEN) and nucleolar organization region (NOR) are indicated. BSXh corresponds to the X-derived heterochromatin accompanying the BS fragment (34). Bar represents 5 µm.
We have used the FISH technique in prometaphase chromosomes of wild-type (Oregon R) males to determine if any of the HeT-A- and TART-positive clones we had obtained derived from the Y chromosome. Three of them did: yw7H7 derives from region h16, yw10F7 derives from region h14 and yw6F11 derives from region h17-h18, the centromeric region of the Y chromosome (Fig. 1A and C). The hybridization signal of yw6F11 occupies only part of the h17-h18 brightly fluorescent block, confirming molecular differences within this region previously suggested by quinacrine staining (34); since we never see the elusive constriction separating h17 from h18, in what follows we will refer to the region recognized by yw6F11 as region h18. The region from which yw6F11 derived is proximal to the HeT-A region previously localized in the h18-h19 junction by Losada et al. (31). We have hybridized sequences from the HeT-A coding region to the YP2D element of T(Y;2)A77p, in which the only Y heterochromatin present is from h17-h18 (29; Fig. 1D). While this probe does not give any hybridization signal, a clear signal is seen when the YAC yw6F11 is used as a probe (Fig. 1B).
The YAC yw6F11 contains 3.1 kb tandem repeat units made of HeT-A and TART 3[prime]-non-coding sequences
During the initial characterization of the 220 kb yw6F11 clone, faint bands corresponding to smaller YACs were observed in the same yeast transformant. This result suggested that the YAC was structurally unstable probably due to the presence of tandemly repeated units in the insert (35). Therefore, the analysis was always carried out using DNA that was prepared from 10 ml cultures grown from single colonies and that was shown not to have additional bands. This clone had another peculiar feature; it was one of the HeT-A 3[prime]-UTR-positive clones that did not show hybridization when further screened with the probe ORF I+II from the coding region of an HeT-A element (the 2.6 kb fragment encompassing nucleotides 824-3500 from the [lambda]23Zn1 clone; accession no. U06920).
To determine the nature of the HeT-A 3[prime]-UTR sequences present in this YAC we used a PCR-based approach. Divergent oligonucleotide primers from the best conserved sequences at the 3[prime]-end of HeT-A elements (HeT-A box region) were used to amplify HeT-A fragments from yw6F11 DNA. The two prevalent fragments amplified following this procedure were cloned into the pGEM-T vector. Clones 6F11p1.3 and 6F11p0.4 contained a 1.3 and a 0.4 kb insert, respectively. To our surprise, sequence analysis of these subclones (Fig. 2A) demonstrated that their HeT-A-related sequences corresponded to those present in the 356 repeat (21), a 3.1 kb tandem repeat originally cloned by Rubin (36).
Figure 2. Analysis of yw6F11 and its physical linkage with Circe sequences. (A) Comparison of the structure of the Y-associated 356 repeat with the sequence of the clones derived from yw6F11. In this diagram the 356 sequence is presented as it was shown in figure 1 of Danilevskaya et al. (21). Numbers indicate the nucleotides in the 356 repeat (accession no. L06099). The actual repeat unit starts at position 2227. The primers used for the PCR amplifications are positioned in the scheme. The 3[prime]-UTR of HeT-A is shown as an open box and the 3[prime]-UTR of TART is shown as a dotted box. The ends of each of the three HeT-A elements are identified by the T6 and TTTAT sequences. (B) Southern blotting hybridization analysis of yw6F11 DNA digested with EcoRI. Autoradiograms show the results of hybridization of four radiolabeled probes, 6F11p1.3, 6F11p0.6, F and Copia, to filter transfers from a single conventional gel. (C) Long-range restriction enzyme analysis of the 18HT repeat cluster. Genomic DNA from Oregon R embryos was digested with the indicated enzymes, electrophoresed through a 0.8% (w/v) agarose gel run in a `waltzer' apparatus for 72 h at 60 V with a 300 s pulse time, blotted to nylon filter and hybridized successively with 6F11p1.3 at 75°C and with probe ORF I+II at 65°C. (D) Genomic DNA from Oregon R embryos was digested with NotI, fractionated on a 1.2% (w/v) agarose gel run at 150 V for 28 h with a pulse time of 80 s and hybridized successively with 6F11p1.3 at 75°C and with pGcirce8 at 65°C. (E) Genomic DNA from imaginal discs and brains of male and female third instar larvae was digested with NotI or XhoI, fractionated on a 0.8% (w/v) agarose gel run at 75 V for 60 h with a pulse time of 240 s and sequentially hybridized with 6F11p1.3 at 75°C and with pGcirce8 at 65°C. Arrows point to the male-specific band.
The reported localization of this repeat in the Y has been subject to several changes, most probably due to the difficulty of achieving sufficiently stringent conditions in experiments of in situ hybridization using tritiated probes. Through in situ hybridization to polytene chromosomes Rubin (36) localized these sequences to the telomeres, where closely related sequences are indeed located; Traverse and Pardue (20) localized these sequences to the pericentric region of the Y; finally, Danilevskaya et al. (21) did not obtain reliable results after in situ hybridization but, by Southern blot analyses, they concluded that these sequences were present distally in both arms of the chromosome BSYy+. FISH experiments with our YAC yw6F11 as hybridization probe have allowed us to unambiguously determine the centromeric origin of the 356 repeat (Fig. 1A and B). In addition, we have also realized that the sequences of this repeat, with no homology to any sequence in the database at the time it was sequenced by Danilevskaya et al. (21), correspond to a 3[prime]-UTR of TART, without its most terminal 3[prime]-end and in the same polar orientation as the HeT-A 3[prime]-UTRs of the repeat (Fig. 2A). Thus, the 356 repeat seems to derive from the amplification of a region from a telomeric chain: three consecutive truncated HeT-A elements (one very tiny) and part of a TART element. To obtain a TART probe for the restriction analysis of the YAC, a 0.6 kb fragment from the TART region of the repeat was PCR-amplified and cloned (clone 6F11p0.6). The relationship between the 356 repeat unit and the clones 6F11p1.3, 6F11p0.4 and 6F11p0.6 is shown in Figure 2A. Thus, clone 6F11p0.4 seems to derive from a 3.1 kb repeat unit with an internal deletion within the TART region.
To determine whether the clone yw6F11 encompassed the genomic cluster of 356 repeats or it was derived from only a part of it, a further analysis of the YAC was carried out. Using PCR with the primer HeT-A box from the conserved sequence at the 3[prime]-end of HeT-A and the primer 1091 from the right (non-centromeric) YAC vector arm, we amplified a 3.8 kb fragment that ends in the middle of a Copia element. This element was found to be inserted in the TART region of the repeat unit, in agreement with the insertion place of a Copia element into this array previously described by Potter et al. (37). To analyze the genomic organization of the 356 repeats included in the YAC, chromosomal DNA isolated from the transformed yeast was digested to completion with EcoRI (which cleaves in the tandem array once per 3.1 kb unit), separated by conventional gel electrophoresis, transferred to a membrane and hybridized with the probes 6F11p1.3 and 6F11p0.6, as well as with probes from the elements F and Copia. The results are shown in Figure 2B. The 6F11p1.3 probe detects multiple bands, but the most intense signal is the 3.1 kb band of the repeat unit. This indicates that most of the repeats of the cluster are cloned in the YAC. The hybridization pattern obtained with this probe does not change when Drosophila genomic DNA is used (data not shown) and coincides with the pattern previously described (20,21). The weaker bands of this pattern can be explained by insertions of transposons like F and Copia (see Fig. 2B) or by deletions within the repeat unit like the one in clone 6F11p0.4.
To avoid confusion with the 356 bp repeat of the 1.688 satellite DNA present on the left arm of the third chromosome (38) we have decided to rename the 356 repeat of Rubin (36); in what follows it will be referred to as the 18HT repeat (h18 HeT-A and TART repeat).
Organization of the heterochromatin near the 18HT repeat cluster
To investigate the physical organization of the 18HT repeat cluster and the neighboring HeT-A sequences we digested DNA from Drosophila embryos (Oregon R) with MluI, NaeI and NotI. Fragments were separated by pulsed-field gel electrophoresis, transferred to a filter and hybridized successively with clone 6F11p1.3 (at high stringency) to detect the 18HT repeats and with probe ORF I+II to detect coding sequences of HeT-A elements (Fig. 2C). MluI cuts in the 18HT repeat but lacks sites in the coding region of HeT-A, NaeI digests in the opposite way and HeT-A elements do not contain internal sites for NotI. Comparison of the hybridization patterns indicates that both probes detect a common NotI DNA fragment of nearly 600 kb (Fig. 2C). Although it is formally difficult to exclude co-migration of separate fragments, this result could be explained by the 18HT repeats being sufficiently close to the decayed HeT-A elements previously found in a contig from the region h18/h19 (31). During the analysis of this contig a tandem array of Circe elements was found between the HeT-As and a tandem of Type I elements. Therefore, the presence of Circe in this NotI fragment could be expected. Figure 2D shows a NotI digest of DNA from embryos probed sequentially with clone 6F11p1.3 (always under high stringency conditions) and with the probe pGcirce8 from the tandem repeat of Circe. Again, one of the NotI fragments detected by the Circe probe corresponds to the fragment detected by 6F11p1.3. Finally, to confirm the Y origin of the ~600 kb NotI fragment, DNA isolated from imaginal discs and brains of male and female third instar larvae was digested with either NotI or XhoI, separated by PFGE, transferred to a membrane and hybridized sequentially with 6F11p1.3 and pGcirce8. The 6F11p1.3 probe recognizes a ~600 kb NotI and a ~200 kb XhoI fragments only in male DNA. The Circe probe detected multiple NotI and XhoI fragments but the ~600 kb NotI fragment that hybridized to both probes was male specific (Fig. 2E).
The occurrence of tandemly repeated sequences in heterochromatin raises problems for mapping genomic DNA. Nevertheless, by combining data from restriction analysis of YAC clones with the results of long-range restriction site mapping of genomic DNA it has been possible to resolve the sequence organization of the centromeric region of the human Y chromosome (39). To apply this mapping strategy it is essential to know if the region under study presents polymorphisms. Figure 3A shows the results of hybridization of the 18HT repeat probe 6F11p1.3 to XhoI, BstBI and BstEII digests of DNAs from Oregon R and Dp(1;f)1187 strains. These enzymes were chosen because they do not cut within the 18HT repeat. The different patterns of restriction fragments seen in each stock show that this region is polymorphic and, therefore, to obtain the physical map it is necessary to analyze the genomic DNA from the Dp(1;f)1187 stock used to construct the YAC library. In this stock the NotI fragment that encompasses the 18HT repeat cluster and the tandem of Circe is slightly longer, 600 kb. This region is also contained in a SfiI fragment of 550 kb. The relative position of Circe in these two fragments was resolved by restriction analysis of the YAC contig from region h18/h19 because the enzymes NotI and SfiI cut distally to the tandem of Circe and very close to each other (Fig. 3D). The long-range restriction site map of the region was constructed by single and double digestions with the infrequently cutting enzymes AscI, PmeI, NotI, SfiI and SgfI. The enzyme AscI was very informative because there was only one AscI site between the 18HT repeat cluster and the AscI sites at the Type I tandem repeats (see Fig. 3D). Thus, the AscI/NotI double digest produces a 350 kb DNA fragment hybridizing with 6F11p1.3 (Fig. 3B). The enzyme PmeI was also informative because it produces an asymmetric cut into the 18HT repeat cluster at the inserted F element (see Fig. 3D). Figure 3C shows that the PmeI digest produces two fragments hybridizing with 6F11p1.3 and that the size of the smaller fragment is reduced in a PmeI/AscI double digest (Fig. 3C). This result and the long-range restriction analysis of the YAC yw6F11 has allowed the orientation of the 18HT repeat cluster on the genomic map to be established (Fig. 3D). The YAC map was constructed using the enzymes XhoI and BstEII, which cut once in the 18HT repeat cluster (see Fig. 3A), and the enzymes ApaI and PmeI, which cut the F element. Thus, the HeT-A and TART sequences in the 18HT cluster have the same orientation as the more distal decayed HeT-A elements, suggesting that both regions derived from rearrangement of a whole telomere and that, after the rearrangement had occurred, a portion of the telomeric retrotransposon chain was amplified. Finally, phosphorimager quantification of the intensity of the bands from several restriction enzyme digests has allowed us to estimate the size of the 18HT cluster at ~100 kb.
Figure 3. Structural organization of the 18HT repeat cluster. (A) Polymorphisms between strains. Genomic DNA from Oregon R (OR R) and Dp(1;f)1187 (Dp1187) strains were digested with the indicated enzymes, electrophoresed through a 1.2% (w/v) agarose gel run in a `waltzer' apparatus for 22 h at 150 V with a 12 s pulse time, transferred to nylon filter and hybridized with 6F11p1.3 at 75°C. (B) Genomic DNA from Dp(1;f)1187 embryos was digested with the indicated enzymes, electrophoresed through a 1% (w/v) agarose gel run at 150 V for 22 h with a 45 s pulse time, blotted to nylon filter and hybridized with 6F11p1.3 at 75°C. (C) Genomic DNA from Dp(1;f)1187 embryos was digested with the indicated enzymes, fractionated on a 1.2% (w/v) agarose gel run at 150 V for 22 h with a 14 s pulse time, transferred to nylon filter and hybridized with 6F11p1.3 at 75°C. (D) Long-range physical map of the centromeric region encompassing the 18HT cluster. The genomic restriction enzyme sites are shown (top). The position of yw6F11 and the YAC contig are drawn underneath. For the enzymes BstEII, XhoI, ApaI and PmeI only sites within the 18HT array and the closest site on either side are shown (bottom). The code used is displayed on the right side of the figure.
The region h18 appears to be conserved in the Y chromosome of D.simulans
Danilevskaya et al. (21) showed that the 18HT sequences were strongly conserved in several D.melanogaster stocks isolated from each other for long periods of time. They interpreted this conservation as a manifestation of the importance of these repeats for the structure of the heterochromatic Y chromosome. We have extended this analysis to the sibling species D.simulans: We have hybridized our probe 6F11p1.3, at high stringency, to NotI digests of DNA from D.simulans embryos and found weak cross-hybridization to a high molecular weight fragment (data not shown). With these data in hand we proceeded to hybridize the YAC yw6F11 to prometaphase chromosomes of larval neuroblasts of D.simulans. The chromosomes were also hybridized with a probe carrying the dodeca satellite of D.melanogaster to mark the centromeric region of the autosomes. Results are presented in Figure 4. yw6F11 mainly recognizes an internal region of the Y. The position of the centromere of the Y chromosome of D.simulans is still ill-defined but the Y locations of the simple satellites correspond well with the locations on the D.melanogaster Y (40). Considering that the Y of D.simulans lacks an NOR region, the position of the signal within this chromosome is rather similar to that of D.melanogaster. Cross-hybridization of such complex sequences from species that have been separated during 2-3 million years suggests that these sequences might confer a selective advantage that is probably related to some essential function.
Figure 4. Cytogenetic localization by FISH of yw6F11 (red) and dodeca satellite (green) in D.simulans. Bar represents 5 µm.
DISCUSSION
The structural analysis of region h18 at the centromere of the Y strongly suggests that this region originated from an ancestral telomere that, once located via unknown mechanisms in an internal position, evolved by regional amplification and multiple transposon insertions, as happens with any typical heterochromatic region. Thus, the 18HT cluster is a satellite DNA that originated by amplification of part of a telomeric chain of HeT-A and TART elements; the region with the decayed HeT-A elements described by Losada et al. (31) would be the result of a transposon invasion into a more proximal region of the original telomere. This view of the HeT-A-homologous sequences in the centromeric region of the Y is not in contradiction with the telomere specificity of HeT-A elements. The parts of the HeT-A elements amplified in the 18HT satellite correspond to the 3[prime]-non-coding imperfect repeats thought by Pardue et al. (41) to play an important role in telomere structure; a similar role could be played by these sequences in establishing the structure of the Y centromere.
Is it possible to find similarities between the apparently different 18HT satellite and dodeca satellite? We believe the answer is yes: they both share similarities with telomerase-generated repeats. To explain the similarity of the dodeca satellite to repeated sequences in different species, Abad et al. (14) suggested that the dodeca satellite might have arisen from widespread telomeric sequences. Dodeca satellite repeats have an asymmetric distribution of guanine and cytosine residues resulting in one strand being relatively G-rich. A similar G/C distribution is shown by the telomerase-generated repeats and in both cases the purine-rich strand is capable of forming intramolecular fold-back structures (42,43). On the other hand, Danilevskaya et al. (44) have realized that the imperfect repeats extending through the 3[prime]-non-coding region of HeT-A elements show a strong strand asymmetry resulting in one strand being A-rich. They also noticed that strong strand asymmetry is a characteristic of the telomerase-generated repeats. From our own analysis of the 18HT satellite we have noticed that the T-rich strand of the imperfect repeats contains short runs of guanine residues and it might be significant that this strand is oriented with the 3[prime]-end towards the end of the chromosome, like the GT-rich strand of typical telomerase-generated DNA. In addition, oligonucleotides having these short contiguous stretches of dG are also capable of forming in vitro G-quartet structures (45). Therefore, these observations tempt us to suggest that the 3[prime] repeat region of HeT-A may structurally behave as the telomeric repeats common to a majority of eukaryotes.
Recent molecular studies in Caenorhabditis elegans (46) and in S.pombe (47) suggest that the telomeric repeats at the ends of the chromosomes are sufficient for spindle attachment in meiotic prophase; therefore, the telomeric repeats can be directly involved in centromeric functions. Although it is still not possible to fully prove that 18HT sequences are sufficient for centromeric function, all the data lead us to propose that the centromere of the Y of D.melanogaster could have arisen through the amplification of telomeric sequences that, before amplification and while placed in their original terminal location, might already have had microtubule binding abilities; these abilities may have later been simply enhanced during evolution in their new location. This obviously does not imply that all centromeres should derive from telomeres. Evolution might have provided many centromeres with sequences that are different from telomere-related sequences but with hidden structural similarities.
ACKNOWLEDGEMENTS
We are grateful to S. Campuzano, M. Berloco, O. Danilevskaya and R. Levis for providing us with transposable element clones. We thank C. Tyler-Smith, J. Avila and D. Lindsley for critical comments and suggestions on the manuscript. We also thank C. Goday for allowing us to use the CCD camera. We thank G. Giovinazzo, L. Fanti and E. Marchetti for instructing M.A. in the FISH technique. M.A. and A.L. were supported by FPI fellowships from the Ministerio de Educación y Cultura. This work was supported by grant PB95-0085 from Dirección General de Enseñanza Superior e Investigación Científica and by an institutional grant from Fundación Ramón Areces.
REFERENCES
*To whom correspondence should be addressed. Tel: +34 913974692; Fax: +34 913974799; Email: avillasante{at}cbm.uam.es
Present address:Ana Losada, Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA
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 A. Villasante, J. P. Abad, R. Planello, M. Mendez-Lago, S. E. Celniker, and B. de Pablos Drosophila telomeric retrotransposons derived from an ancestral element that was recruited to replace telomerase Genome Res., December 1, 2007; 17(12): 1909 - 1918. [Abstract] [Full Text] [PDF]
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 E. N. Andreyeva, T. D. Kolesnikova, O. V. Demakova, M. Mendez-Lago, G. V. Pokholkova, E. S. Belyaeva, F. Rossi, P. Dimitri, A. Villasante, and I. F. Zhimulev High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants PNAS, July 31, 2007; 104(31): 12819 - 12824. [Abstract] [Full Text] [PDF]
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A. Villasante, J. P. Abad, and M. Mendez-Lago Centromeres were derived from telomeres during the evolution of the eukaryotic chromosome PNAS, June 19, 2007; 104(25): 10542 - 10547. [Abstract] [Full Text] [PDF]
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 L. Usakin, J. Abad, V. V. Vagin, B. de Pablos, A. Villasante, and V. A. Gvozdev Transcription of the 1.688 Satellite DNA Family Is Under the Control of RNA Interference Machinery in Drosophila melanogaster Ovaries Genetics, June 1, 2007; 176(2): 1343 - 1349. [Abstract] [Full Text] [PDF]
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H. Biessmann, S. Prasad, V. F. Semeshin, E. N. Andreyeva, Q. Nguyen, M. F. Walter, and J. M. Mason Two Distinct Domains in Drosophila melanogaster Telomeres Genetics, December 1, 2005; 171(4): 1767 - 1777. [Abstract] [Full Text] [PDF]
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X. Bi, D. Srikanta, L. Fanti, S. Pimpinelli, R. Badugu, R. Kellum, and Y. S. Rong Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance PNAS, October 18, 2005; 102(42): 15167 - 15172. [Abstract] [Full Text] [PDF]
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 J. P. Abad, B. de Pablos, K. Osoegawa, P. J. de Jong, A. Martin-Gallardo, and A. Villasante Genomic Analysis of Drosophila melanogaster Telomeres: Full-length Copies of HeT-A and TART Elements at Telomeres Mol. Biol. Evol., September 1, 2004; 21(9): 1613 - 1619. [Abstract] [Full Text] [PDF]
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J. P. Abad, B. de Pablos, K. Osoegawa, P. J. de Jong, A. Martin-Gallardo, and A. Villasante TAHRE, a Novel Telomeric Retrotransposon from Drosophila melanogaster, Reveals the Origin of Drosophila Telomeres Mol. Biol. Evol., September 1, 2004; 21(9): 1620 - 1624. [Abstract] [Full Text] [PDF]
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K. A. Maggert and K. G. Golic The Y Chromosome of Drosophila melanogaster Exhibits Chromosome-Wide Imprinting Genetics, November 1, 2002; 162(3): 1245 - 1258. [Abstract] [Full Text] [PDF]
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E. Casacuberta and M.-L. Pardue Coevolution of the Telomeric Retrotransposons Across Drosophila Species Genetics, July 1, 2002; 161(3): 1113 - 1124. [Abstract] [Full Text] [PDF]
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W. J. Miller, A. Nagel, J. Bachmann, and L. Bachmann Evolutionary Dynamics of the SGM Transposon Family in the Drosophila obscura Species Group Mol. Biol. Evol., November 1, 2000; 17(11): 1597 - 1609. [Abstract] [Full Text] [PDF]
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