Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster
© Cuadrado and Jouve; licensee BioMed Central Ltd. 2011
Received: 3 February 2011
Accepted: 26 April 2011
Published: 26 April 2011
In recent years, substantial progress has been made in understanding the organization of sequences in heterochromatin regions containing single-copy genes and transposable elements. However, the sequence and organization of tandem repeat DNA sequences, which are by far the majority fraction of D. melanogaster heterochromatin, are little understood.
This paper reports that the heterochromatin, as well as containing long tandem arrays of pentanucleotide satellites (AAGAG, AAGAC, AATAT, AATAC and AACAC), is also enriched in other simple sequence repeats (SSRs) such as A, AC, AG, AAG, ACT, GATA and GACA. Non-denaturing FISH (ND-FISH) showed these SSRs to localize to the chromocentre of polytene chromosomes, and was used to map them on mitotic chromosomes. Different distributions were detected ranging from single heterochromatic clusters to complex combinations on different chromosomes. ND-FISH performed on extended DNA fibres, along with Southern blotting, showed the complex organization of these heterochromatin sequences in long tracts, and revealed subclusters of SSRs (several kilobase in length) flanked by other DNA sequences. The chromosomal characterization of C, AAC, AGG, AAT, CCG, ACG, AGC, ATC and ACC provided further detailed information on the SSR content of D. melanogaster at the whole genome level.
These data clearly show the variation in the abundance of different SSR motifs and reveal their non-random distribution within and between chromosomes. The greater representation of certain SSRs in D. melanogaster heterochromatin suggests that its complexity may be greater than previously thought.
One of the most enigmatic aspects of genome organization in multicellular eukaryotes is the regionalization of chromosomes into euchromatin and heterochromatin domains. Heterochromatin, originally named "junk DNA" because no coding function could be found for it, is now considered essential for the epigenetic maintenance of centromeric function as well as for other cellular, developmental and evolutionary processes [1–3]. In most eukaryotes, the main components of heterochromatin are families of highly tandem repeated DNA or satellite DNA organized as multiple copies of a monomer sequence arranged in a head to tail pattern over megabase-long arrays. Running from a few base pairs to more than 1 kb in length, repeated units show a wide range of sizes and complexity. Despite sequence divergence between monomers of the same family often being very low over long arrays, satellite DNA in heterochromatin can change rapidly in nucleotide sequence or copy number during evolution. As a consequence, large numbers of unrelated satellite DNA families commonly compound the profile of satellite DNA in genomes [4, 5].
Drosophila melanogaster provides a model for studies of heterochromatin. About 59 Mb of the 176 Mb female genome, including the proximal half of the X chromosome, the pericentromeric region of autosomes 2 and 3, and most of the dot-like chromosome 4 is heterochromatic. The entire male Y chromosome (41 Mb) is also heterochromatic . However, polytene chromosomes, which have proved useful in mapping euchromatin regions, provide minimal resolution in heterochromatin analyses. Due to its late replication, the heterochromatin remains immersed in the diffuse and unbanded chromocentre region. Thus, mitotic rather than polytene chromosomes are preferable for chromosome mapping of heterochromatic sequences .
Over the last three decades, different families of highly repeated tandem sequences have been molecularly characterized and physically mapped to the chromosomes of D. melanogaster using different cloning strategies. Most of the highly repeated families of the fruit fly genome are composed of short repeat units 5-12 bp long, arrayed in tandem and extended over several megabases of DNA. These highly repeated sequences are localized in specific segments of the heterochromatin, which may contain different sets of satellite DNA [8–11].
The goal of large-scale genome projects is to convert the initial draft of euchromatic sequences into a complete telomere-to-telomere sequence for each chromosome. The Drosophila Heterochromatin Genome Project has made substantial progress in identifying contiguous sequences of the heterochromatin regions containing single-copy genes and dense clusters of transposable elements. However, the sequence and organization of the highly tandem repetitive DNA fraction - the vast majority of D. melanogaster heterochromatin - is little known . The apparent absence of identified highly repeated sequences in certain heterochromatic regions and BACs covering the extensive heterochromatin gaps, suggests that unknown classes of highly repetitive DNAs must be present in this fraction of the D. melanogaster chromosomes. To determine what they are will require new technologies .
Short motifs of 1-6 bp repeated in tandem are classified as SSRs or microsatellites [14, 15]. Although the genomes of higher organisms contain some long microsatellites (up to 500 nucleotides), in general these polymorphic loci are no longer than 100 nucleotides. However, in many species, SSRs may also be organized into long stretches of nearly 100 to several thousand tandem units mainly clustered in the heterochromatin, also referred to as satellite SSRs [16, 17]. For example, in many vertebrate species, the heterochromatic sex chromosomes are rich in clusters of GATA and GACA repeats . Long arrays of AAGAG and AATAT are found in the heterochromatin of fruit fly chromosomes . Moreover, GGAAT and CATTT repeats have been found in the satellite regions of human chromosomes . There is some evidence that the origin and evolution of satellite families, including those with relatively complex and longer repeated units, can be explained by the autoreplicative properties of different SSRs .
In the present study, a recently reported, simple and highly efficient non-denaturing FISH method (ND-FISH) was used to localize the classic satellites (AAGAG, AAGAC, AATAT and AATAC) and AACAC repeats . Heterochromatic regions enriched in SSRs were also sought, and the physical distribution of 16 different SSR types (mono-, di-, tri- and tetranucleotide repeats) studied in both polytene and mitotic chromosomes. An ND-FISH analysis employing DNA fibres was also performed and the SSRs characterized by Southern hybridization.
Mitotic and polytene chromosomes of D. melanogaster were obtained using third instar larvae of a wild type strain kept in our laboratory for about 20 years. The brains and salivary glands of individual larvae were dissected out in 45% acetic acid before squashing on a clean microscope slide with a drop of 45% acetic acid. After removing the cover slips by freezing, the slides were air dried.
DNA fibres were obtained from disrupted nuclei following a derivative of the alkaline/ethanol method described by Fidlerová et al. . The brains of third instar larvae were dissected in a drop of water. Neuroblast tissues was disaggregated with a pestle in a drop of 3:1 ethanol:acetic acid on one end of a clean microscope slide. The resulting cell suspension was allowed to air-dry. The slides were then rinsed in 100 ml of phosphate-buffered saline solution (PBS) in a Coplin jar for 1 min. After removing the PBS solution (but not allowing the slide to dry out), the cell-containing area was incubated with 100 μl of lysing buffer (0.5 M NaOH/30% ETOH) for 1 min to disrupt the nuclei before stretching the fibres by tilting the slide at 45° and letting the buffer run away. 100 μl of 100% ethanol were then added to the cell-containing area before post-fixation employing an ethanol series (70%, 100%) (performed in a Coplin jar), allowing 5 min in each solution. Finally, the slides were air-dried.
Probes and labelling
Oligodeoxyribonucleotides used as probes to analyze SSRs in the present study and references of works where the distribution of SSRs has been reported previously by in situ hybridization in D. melanogaster chromosomes.
Probes used in study
AAC; AAG; AAT, AAC; AGC; ACT; AGC; AGG; ATC; CCG
(AAC)5; (AAG)5; (AAT)5; (ACC)5; (ACG)5; (ACT)5; (AGC)5; (AGG)5; (ATC)5; (CCG)5
AAGAC; AAGAG; AATAC; AATAT
(AAGAC)3; (AAGAG)3; (AATAC)3; (AATAT)3
Non-denaturing fluorescence in situ hybridization
ND-FISH was performed as previously described . Briefly, preparations were incubated at 24°C in a humidity chamber for 2 h with 30 μl of hybridization buffer containing 2 pm of the probe in 2×SSC. For post-washing, slides were immersed in 4×SSC/0.2% Tween20 and agitated for 10 min at room temperature (RT). The detection of digoxigenin and biotin was respectively undertaken by incubating the slides in fluoresceinated anti-digoxigenin (Roche Applied Science) or streptavidin-Cy3 (Sigma) in 5% (w/v) BSA for 1 h at 37°C. Before staining the DNA with DAPI, the slides were rinsed for 10 min in 4×SSC/0.2% Tween20 at RT. The slides were then mounted in antifade solution (Vector Laboratories).
Fluorescence microscopy and imaging
Slides were examined using a Zeiss Axiophot epifluorescence microscope. For each motifs we analysed at least 10 cells. The images from the biotin/Cy3, digoxigenin/Fitc and DAPI staining procedures were recorded separately using a cooled CCD camera (Nikon DS). The exposure times were variable depending of the intensity of the in situ signals obtained with each probe. The localization of the signals relative to the DAPI banding pattern was resolved by merging digital images using Adobe Photoshop. The length of single DNA fibres was measured taking into account that a beaded, interrupted signal often denotes a maximally decondensed DNA fibre. An average of 3 Kb μm-1 is reported for mammalian and Drosophila DNA [23, 24].
Southern blot analyses
Genomic DNA was isolated from adult flies using the DNeasy Blood & Tissue kit (Qiagen). DNA from both males and females (7.5 μg) was digested with Alu I, Rsa I and Hinf I restriction endonucleases, divided into three aliquots and separated on 1% agarose gel. After electrophoresis the gels were blotted onto nylon membranes (Biodyne, Pall Corporation) under alkaline conditions following standard techniques. Each membrane was cut into three and hybridized successively with different digoxigenin-labelled oligonucleotides as probes. To ensure high stringency, the temperature of hybridization and the post-hybridization washes were estimated for each oligonucleotide probe according to the Tm and adhering to the GC content rule: 30.7°C for AATAC3, 36.2°C for ACT5, AAG5 and AAC5, 38.9°C for AAGAG3, AACAC3 and AAGAC3, 44°C for GATA5, and 54.3°C for AG10, AC10 and GACA5. Hybridization was performed overnight using 2 pm of oligonucleotides in 5×SSC, 0.02% SDS, 0.01% LSS and 0.3% blocking reagent (Roche). Filters were washed with 0.15×SSC and 0.1% SDS at the corresponding Tm for 30 min before detection using anti-digoxigenin-AP, Fab fragments (Roche), and CDP-Star (Roche) according to the manufacturer's recommendations. The techniques followed were basically as described in Loarce et al. .
Chromosomal localization of pentanucleotide DNA satellites by ND-FISH
The AAGAG probe was found at different intensities in multiple clusters in the chromocentre of polytene chromosomes and in interphase neuroblast nuclei (Figure 1a-c and 1e). All mitotic chromosomes showed in situ signals (Figure 1b and 1e). The number of clusters on chromosome 2 could not be determined due to the proximity of their signals, which were arranged in a dispersed pattern along most of the heterochromatin (het) of the right arm (2Rhet). The multiple sites observed on the Y chromosome were concentrated in three regions along its length, with two on YL and one on YS. AAGAG repeats were also found in significant amounts on 3Rhet and in two minor clusters, one distal on XR and the other on chromosome 4.
(AAGAC)3 showed a similar chromosomal distribution to (AAGAG)3. However, when two-colour ND-FISH was performed using both probes simultaneously, differences were observed between the polytene and mitotic chromosomes (Figure 1c and 1e). In male metaphases, as with the AAGAG probe, multiple sites were observed on the Y chromosome, concentrated in the same regions (Figure 1e). The AAGAC repeats were localized in a strong cluster on 2Rhet and in a minor site on 3Rhet (Figure 1d).
The AATAT probe was also found in the chromocentre of polytene chromosomes (Figure 1f). A detailed analysis allowed the AATAT repeats to be localized precisely at the base of the polytenized regions of chromosomes 3R and 4 (Figure 1g). In mitotic chromosomes these repeats were primarily found on chromosome 4. In addition, multiple sites of lower intensity were detected on chromosome Y (the strongest at the two tips). Two minor clusters were also seen, one distal on XR and the other on 3R, presumably near the euchromatin-heterochromatin junction (Figure 1h).
Finally, a few AACAC signals were found dispersed in the chromocentre (Figure 1m), in the unbanded regions of chromosome arm 2R (Figure 1n). In metaphase chromosomes these repeats were situated on 2Rhet and YS (Figure 1o).
Chromosomal localization of mono- and dinucleotide SSRs by ND-FISH
The A20 probe showed a great number of signals in polytene chromosomes. A high density of A repeats was seen on the X chromosome, especially concentrated near the chromocentre (Figure 2a), as previously reported (Table 1, ). In mitotic chromosomes, A20 was found interstitially along chromosome arm XL and in three clusters on chromosome Y, with two on the long arm and one in its satellite (Figure 2c). A disperse distribution of A signals was observed throughout the euchromatin in mitotic chromosomes when using long image capturing exposure times. The highest concentration was seen on the distal half of chromosome X, contrasting with the absence of signals on chromosome 4. In addition, higher concentrations of dispersed signals were observed in the proximal chromosome arm 2R (Figure 2b).
Polytene chromosomes hybridized weakly with the C probe. However, with the help of the CCD camera, these repeats were observed as many bands that decreased in number towards the chromocentre, and which were particularly concentrated on the X chromosome (Figure 2d).
With the exception of chromosome 4, a large number of intense signals for (AC)10 were seen along the length of the polytene chromosomes arms. As described above for the mononucleotide repeats, the density of signal along the X chromosome was higher. In addition, a few signals of different intensity were found dispersed in the chromocentre, some in the unbanded regions of chromosome 2 (Figure 2e). In mitotic chromosomes, weak signals were seen dispersed over most of the euchromatin. A cluster of stronger signals was seen near the centromere of chromosome arm 2R (Figure 2f).
The distribution and intensity of the (AG)10 signals fit the general pattern described for AC repeats in polytene chromosomes (Figure 2g-h). In addition, the AG probe was found in multiple clusters of different intensity in the chromocentre. The unbanded region of chromosome 2 was particularly rich (Figure 2g-h). AG repeats were detected in multiple clusters in all mitotic chromosomes. Several sites were observed on chromosome 2, especially concentrated on 2Rhet (Figure 2j), and in the two tips of chromosome Y. A cluster was also found on 3Rhet along with two minor sites on chromosome 4 and another on the tip of chromosome XR (Figure 2i).
Chromosomal localization of tri- and tetranucleotide SSRs by ND-FISH
Increasing the CCD camera exposure time revealed specific patterns of low intensity signals for AAC, ACC, ATC, ACG, AGC, AGG and CCG distributed as a large number of signals along the length of the polytene arms (Figure 3i and j). Regardless of differences in the abundance and localization of each motif on the polytene chromosomes, a uniform pattern of distribution was observed over all chromosome arms, but particularly on chromosome X. The use of very long exposure times revealed a characteristic dispersion pattern of signals in mitotic chromosomes (Figure 3k).
ND-FISH was used in previous work to determine the distribution of (GACA)5 and (GATA)5 in polytene chromosomes of D. melanogaster ( and Figure 3l and 3o respectively). That analysis was here extended by analysing mitotic chromosomes (Figure 3m-n and 3p-q respectively). In addition to the high concentration of GACA repeats found in the chromocentre of polytene chromosomes, a rich pattern of signals of similar intensity was seen over the chromosome arms (Figure 3l). The strongest (GATA)5 signals were detected at the chromocentre and a few specific sites on the chromosomes arms (Figure 3o). GACA repeats were especially numerous on mitotic chromosomes 2Rhet and Y (two clusters on YL and one site on its satellite). A minor cluster was also detected on chromosome 3Rhet. The strongest GATA signals were seen on the long arm of chromosome X, with minor sites on chromosomes 2, 4 and Y (Figure 3p-q).
Location of SSRs relative to cytogenetic heterochromatic regions
The detailed analysis of polytene chromosomes allowed the SSRs clustered at the boundaries of the pericentromeric heterochromatin to be assigned to different heterochromatin segments. For example the AATAT repeats found near the chromocentre on 3R might correspond to the signal found on 3Rhet near the frontier between the euchromatin and heterochromatin, presumably at the boundary of region h58 (Figure 1f). In the same way, the GATA repeats found near the chromocentre at the base of chromosome X might correspond to the signals clustered on the long arm of chromosome X at the heterochromatin-euchromatin border in the region h26 (Figures 3o).
Analysis of DNA fibres by ND-FISH
Molecular organization of SSRs in D. melanogaster
Initially our Southern results show the abundance and size of the fragments to vary significantly between different SSRs. Only the pentanucleotide probes hybridized to the restriction enzyme-resistant DNA (Figure 6B-E), indicating the absence of the restriction sites and the existence of large arrays. Three noticeable differences were detected among the probes. (AAGAG)3 gave the strongest hybridization pattern with the genomic DNA of both sexes, with most of the fragments longer than 20 kb (Figure 6B), while a prominent male-specific hybridization pattern was observed with the (AAGAC)3 and (AATAC)3 probes (Figure 6C-D), defining the existence of the Y chromosome. Finally (AACAC)3 revealed multiple and well defined bands of hybridization ranging in size from 1 to 20 kb.
The remaining SSR probes returned specific patterns of hybridization varying in number and fragment size. The (AC)10 probe showed the strongest hybridization, with fragments ranging from a few base pairs to 5 kb in size (Figure 6F). (AG)10, (AAG)5, (AAC)5, (ACT)5 (GATA)5 and (GACA)5 showed specific patterns with wide spectrums of well-defined fragments ranging from 0.1 to 4 kb in size (Figure 6G-K respectively).
The remarkable differences between the motifs and the composite SSRs, for example AG and AAG and the composite AAGAG or AC and AG and the composite GACA, revealed by southern pattern (Figure 6), provide evidence for the specific amplification of each SSRs at particular chromosomal locations.
Reliability of the ND-FISH technique in the visualisation of SSR-enriched chromosome regions
The five pentanucleotides SSRs used to analyze the classic DNA satellites of D. melanogaster differ from one another by at least one nucleotide. Therefore, it is to be expected that these probes might hybridize with one another under low stringency hybridization conditions. The contrasting distribution results for these SSRs show, however, that even probes with 80% identity provide motif-specific hybridization patterns in ND-FISH. Moreover, except for two faint additional signals with (AATAC)3 and (AAGAC)3, the results obtained here were in complete agreement with previous localizations involving clones containing the satellite repeats as probes in high stringency in situ hybridization experiments using other flies stocks ([10, 11] and Figures 1 and 4). These results suggest that heterochromatic SSRs clusters are conserved among strains. However we can no discard certain level of polymorphism (extents/distributions of SSRs) among different flies stocks. Additional clusters of AG and AC on mitotic chromosomes were detected beyond those discovered in previous work involving FISH and long duplex probes composed of simple motifs . This result indicates that ND-FISH is a higher efficient technique improving the sensitivity of detection of SSRs enriched chromosome regions.
No attempt was made to construct a higher resolution physical map of SSR clusters in the heterochromatin. Even when analysing the heterochromatic regions in well spread prometaphase cells, it was impossible to reliably assign probes to specific blocks in DAPI-stained chromosomes (since there are about 100 Mb of heterochromatin divided into 61 bands) (e.g., see Figure 1b and 2i). Further, due to the fluorescence spreading, it could not be determined whether strong signals were actually produced by two or more neighbouring SSR clusters. Nevertheless, Figure 4 provides a useful working map of the highly repetitive components of the D. melanogaster genome, which further analyses ought to improve (e.g., using simultaneous and/or sequential ND-FISH with different probes and analysing ordered chromosome rearrangements or chromosomes with deletions of specific heterochromatic bands, etc.).
The ND-FISH technique is simpler, faster and more efficient than any previously reported in situ method for analyzing the chromosome distribution of SSRs . Another advantage of ND-FISH over standard FISH is that it avoids chromosome denaturation; thus, the morphology of the chromosomes is well maintained. This is very important when successive reprobing is required on the same slide. Moreover, ND-FISH probably prevents DNA losses. This is desirable for higher resolution mapping using DNA fibres, which often become fragmented during the denaturing step of standard FISH. Thus, ND-FISH resolves one of the main limitations of the analysis of DNA fibres by conventional FISH. The results in Figure 5 show that neuroblasts are an excellent source of nuclei for lysing, and that oligonucleotides used as probes in ND-FISH permit the mapping of SSRs in extended chromatin fibres.
D. melanogaster heterochromatin is enriched in SSRs
D. melanogaster is a model for heterochromatin studies and the euchromatin-heterochromatin boundaries have been fully analysed. Moreover, heterochromatic sequences of about 20 Mb, mainly containing single-copy sequences and transposable elements, have been almost completely mapped [13, 28]. However, as in other "finished" genomes, the sequence and structure of the main components of the heterochromatin, which contains highly repetitive tandem sequences, are poorly characterized .
As far as we know, the only SSRs molecularly characterized with a unit length under 4 bp in the heterochromatin of D. melanogaster are TAA and GATA. TAA is involved in a complex sequence of 5.9 kb which maps to h26, the most distal division of the cytogenetic map . The present data show a distribution of AAT very close to the proximal region of chromosome X (Figure 3h), suggesting it might be the same sequence array.
Singh et al.  reported the existence of GATA repeats concentrated in the proximal region of the X chromosome as a minor repeat class in the genome of D. melanogaster. The sequencing of chromosome X showed GATA to also be present in the pericentromeric heterochromatin ends while the most proximal sequence scaffold begin also in TAGA repeats, flanking a gap of unknown size in the heterochromatic region h26 . The present ND-FISH data suggest that the stronger signals found with the (GATA)5 probe at the euchromatin-heterochromatin boundary of the X chromosome (Figure 3o-q) might represent this unsequenced gap.
Structure of SSRs clusters
Satellite DNA can be extended over megabases of DNA but the maximum length is unknown due to the lack of large DNA clones containing highly repeated sequences. For example, two large blocks of AATAT and AAGAG satellites ranging up to approximately 380 kb, interrupted by five transposons, have been identified in the functional centromeric region of the minichromosome Dp1187 in D. melanogaster, but only contigs of about 8 kb of these repeats have been obtained [32, 33].
Several kilobases of target sequence are required to produce consistent hybridization signals in metaphase chromosomes, although this does not mean that the target must exist in long interrupted stretches. On the other hand, no important differences were found between the probes here analysed for visualising the arrays of SSRs using DNA fibres (Figure 5). The characteristic appearance of a beaded rather than a continuous fluorescent track limits the estimation of the number of repeats in the continuous tandem. Thus, in addition to the gaps observed, suggests several kilobases of unrelated sequence flanked by SSRs, the existence of several DNA kilobases of unrelated sequences within the fluorescence string of signals cannot be ruled out (Figure 5f).
Clear differences were observed after Southern blot hybridization. The pentanucleotides, found exclusively in the heterochromatin by ND-FISH, hybridized at the exclusion limit of the gel, indicating the existence of large arrays of these repeats in the enzyme-resistant DNA fraction (Figure 6B-E). In addition, the specific pattern of multiple fragments observed over a wide range of molecular weight probably reflects heterogeneity within the tandem repeats and/or the existence of other sequences present within these highly repetitive blocks. This agrees with the analysis of sequences of individual pentanucleotide SSR tracts, demonstrating that they could be imperfect and might be flanked by DNA sequences, mainly related to transposable elements [8, 33].
None of the SSRs with a repeat motif of less than four bases analysed by Southern blotting hybridized with the undigested DNA fraction, and no fragments with a molecular weight of over 10 kb were found (Figure 6F-K). This suggests that these SSRs must be organised in clusters of short tandem repeat stretches instead of the longer blocks that characterize the classic satellites. However, they provide the extended regions of several kilobases required to produce consistent in situ signals on mitotic chromosomes. The differences in genomic organisation suggested by the Southern blot analysis might explain why the novel SSRs clustered in the heterochromatin of D. melanogaster, have never been previously isolated or cloned using the satellite DNA fragments obtained by ultracentrifugation in caesium density gradients .
Abundance and distribution of SSRs in D. melanogaster chromosomes
The analysis of complete euchromatin sequences revealed that, as for most of the genomes examined to date, the D. melanogaster genome shows great variation in terms of the abundance and distribution of SSRs [34–37]. The present results for the polytene chromosomes agree with some of the above database-held information. For example, in the euchromatic chromosome arms, the SSRs based on the dinucleotides AC and AG were the most common, followed by the SSR with the mononucleotide A. In general, trinucleotide repeats were only detected in small amounts by ND-FISH (Figure 3a-k). These results might be explained by the size of the SSR loci and the abundance of the different motifs found in D. melanogaster genome. The frequency of the complete trinucleotide SSR set was found to be about half that of the AC repeat loci. Moreover, about 10% of the AC repeat loci have more than 100 bp while trinucleotide SSRs loci with more than 50 bp are very rare and only the ACT and AAT repeats have more than 100 bp . Therefore, a minimum target sequence size (long SSRs or high density of short loci) is required for ND-FISH signals to be detected even when using polytene chromosomes. As expected, because long pentanucleotide SSR loci are rare in D. melanogaster sequenced genome they were mainly localized in the chromocentre of polytene chromosomes (Figure 1). However, half of the SSRs scanned in this work, some of which were abundant in the heterochromatin, also had a significant presence in the euchromatin (Figure 2 and 3).
The present results show an uneven chromosome distribution for several motifs within and between the individual chromosomes of D. melanogaster. These motifs show a heterogeneous distribution with marked differences in density, both in the euchromatin and heterochromatin. This confirms earlier observations made by in situ hybridization showing a high density of SSRs, especially for the mono- and dinucleotide repeats in the euchromatic arm of chromosome X and their absence in chromosome 4, to correlate with the phenomena of gene dosage compensation and recombination [39, 40]. The results of the present work suggest that both the absolute and relative frequencies of SSRs recorded to date in the D. melanogaster database would be significantly different if the whole-genome sequence were available. The high quantity of GATA repeats in the heterochromatin with respect to the euchromatic regions supports this assumption (Figure 3o-q). Moreover, with the exception of GATA repeats, the remaining SSRs were less abundant in the X than in the Y chromosome. The differences between sexes are notable with respect to some repeats such as AAGAG or AAGAC, which are particularly enriched in the Y chromosome (compare Figure 1b and 1e). Previous molecular characterization of SSRs using total genomic DNA from embryos of D. melanogaster indicated that AAGAG and AATAC comprise 5.6%, and 0.52% respectively of the genome . The present ND-FISH results indicate that some SSRs motifs are very abundant in the heterochromatin of D. melanogaster, with AAGAG and AATAC the most and least common respectively.
Our ND-FISH method makes highly efficient the detection of SSRs in D. melanogaster chromosomes. The data clearly show the variation in the abundance of different SSR motifs and reveal their non-random distribution within and between chromosomes. As judged by our results, the greater representation of certain SSRs in D. melanogaster heterochromatin suggests that its complexity may be greater than previously thought. Thus, the possibility that these sequences may be implicated in some putative heterochromatin role(s) cannot be excluded.
This study was supported by grants from the Spanish Ministry of Science and Innovation (AGL 2009-10373). The authors thank Adrian Burton for linguistic assistance.
- Grewal SI, Elgin SC: Heterochromatin: new possibilities for the inherence of structure. Curr Opin Genet Dev. 2002, 12: 178-187.PubMedView ArticleGoogle Scholar
- Morris CA, Moazed D: Centromere assembly and propagation. Cell. 2007, 128 (4): 647-650.PubMedView ArticleGoogle Scholar
- Martienssen RA: Maintenance of heterochromatin by RNA interference of tandem repeats. Nat Genet. 2007, 35 (3): 213-214.View ArticleGoogle Scholar
- Bosco G, Campbell P, Leiva-Neto JT, Markow TA: Analysis of Drosophila species genome size and satellite DNA content reveals significant differences among strains as well as between species. Genetics. 2007, 177: 1277-1290.PubMed CentralPubMedView ArticleGoogle Scholar
- Plohl M, Luchetti A, Mestrovic N, Mantovani B: Satellite DNAs between selfishness and functionality: Structure, genomics and evolution of tandem repeats in centromeric (hetero)chromatin. Gene. 2008, 409: 72-82.PubMedView ArticleGoogle Scholar
- Hoskins , et al: Heterochromatic sequences in a Drosophila whole-genome shotgun assembly. Genome Biology. 2002, 3: Research0085.1-16.View ArticleGoogle Scholar
- Zhang P, Spradling AC: The drosophila salivary gland chromocenter contains highly polytenized subdomains of mitotic heterochromatin. Genetics. 1995, 2: 659-670.Google Scholar
- Lohe AR, Brutlag DL: Multiplicity of satellite DNA sequence in Drosophila melanogaster. Proc Natl Acad Sci USA. 1986, 83: 696-700.PubMed CentralPubMedView ArticleGoogle Scholar
- Abad JP, Carmena M, Baars S, Saunders RDC, Glover DM, Ludeña P, Sentis C, Tyler-Smith C, Villasante A: Dodeca satellite: A conserved G+C rich satellite from the centromeric heterochromatin of Drosoohila melanogaster. Proc Natl Acad Sci USA. 1992, 89: 4663-4667.PubMed CentralPubMedView ArticleGoogle Scholar
- Lohe AR, Hilliker AJ, Roberts PA: Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics. 1993, 134: 1149-1174.PubMed CentralPubMedGoogle Scholar
- Makunin IV, Pokholkoa GV, Kholodilow NG, Zakharkin SO, Bonaccorsi S, Dimitri P, Zhimulev IF: A novel simple satellite DNA is colocalized with the Stalker retrotransposon in Drosophila melanogaster heterochromatin. Mol Gen Genen. 1999, 261: 381-387.View ArticleGoogle Scholar
- Celniker SE, et al: Finishing a whole-genome shotgun: Release 3 of the Drosophila melanogaster euchromatic genome sequence. Genome Biol. 2002, 3: Research00791-14.View ArticleGoogle Scholar
- Hoskins RA, et al: Sequence finishing and mapping of Drosophila melanogaster heterochromatin. Science. 2007, 16: 1625-1628.View ArticleGoogle Scholar
- Tautz D, Renz M: Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res. 1984, 4127-4138.Google Scholar
- Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatelite' regions in human DNA. Nature. 1985, 314: 67-73.PubMedView ArticleGoogle Scholar
- Schäfer R, Ali S, Epplen JT: The organization of the evolutionarily conserved GATA/GACA repeats in the mouse genome. Chromosoma. 1986, 93: 502-510.PubMedView ArticleGoogle Scholar
- Cuadrado A, Schwarzacher T: The chromosomal organization of simple sequence repeats in wheat and rye genomes. Chromosoma. 1999, 107: 587-594.View ArticleGoogle Scholar
- Epplen JT: On simple repeated GAT/CA sequences in animal genomes: A critical reappraisal. Journal of Heredity. 1988, 79: 409-417.PubMedGoogle Scholar
- Guy J, Spallluto C, McMurray A, Hearn T, et al: Genomic sequence and transcriptional profile of the boundary between pericentromere satellites and genes on human chromosome arm 10p. H Mol Genet. 2000, 9 (13): 2029-2042.View ArticleGoogle Scholar
- Hancock JM: Genome size and the accumulation of simple sequence repeats: implications of new data from genome sequencing projects. Genetica. 2002, 115: 93-103.PubMedView ArticleGoogle Scholar
- Cuadrado N, Jouve N: Chromosomal detection of simple sequence repeats (SSRs) using nondenaturing FISH (ND-FISH). Chromosoma. 2010, 19: 495-503.View ArticleGoogle Scholar
- Fidlerová H, Senger G, Kost M, Sanséau P, Sherr D: Two simple procedures for releasing chromatin from routinely fixed cells for fluorescence in situ hybrization. Cytogenet Cell Genet. 1994, 65: 203-2005.PubMedView ArticleGoogle Scholar
- Wiegant J, Kalle W, Mullenders L, Brookes S, Hoovers JMN, Dauwerse JG, van Ommen GJB, Raap AK: High-resolution in situ hybridization using DNA halo preparations. Hum Mol Genet. 1992, 1: 587-591.PubMedView ArticleGoogle Scholar
- Kuhn GCS, Teo CH, Schwarzacher T, Heslop-Harrison JS: Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonomologous satellites DNAs in cactophilic Drosophila species. Heredity. 2009, 102: 453-464.PubMedView ArticleGoogle Scholar
- Loarce Y, Hueros G, Ferrer E: A molecular linkage map of rye. Theor Appl Genet. 1996, 93: 1112-1118.PubMedView ArticleGoogle Scholar
- Pimpinelli S, Berloco M, Fanti L, Dimitri P, Bonaccorsi S, Marchetti E, Caizzi R, Caggese C, Gatti M: Transposable elements are stable structural components of Drosophila melanogaster heterochromatin. Proc Natl Acad Sci USA. 1995, 92: 3804-3808.PubMed CentralPubMedView ArticleGoogle Scholar
- Cuadrado A, Jouve N: Similarities in the chromosomal distribution of AG and AC repeats within and between Drosophila, human and barley chromosomes. Cytogenet Genome Res. 2007, 119: 91-99.PubMedView ArticleGoogle Scholar
- Smith CD, Shy S, Mungall CJ, Karpen GH: The release 5.1 annotation of Drosophila melanogaster heterochromatin. Science. 2007, 316: 1586-1591.PubMed CentralPubMedView ArticleGoogle Scholar
- Mardis e, McPherson J, Martienssen R, Wolson R, McCombie WR: What is finished, and why does it matter. Genome Research. 2002, 12: 669-671.PubMedView ArticleGoogle Scholar
- O'Hare K, Chadwick BP, Constantinou A, Davis Aj, Mitchelson A, Tudor M: A 5,9-kb tandem repeat at the euchromatin-heterochromatin boundary of the X chromosome of Drosophila melanogaster. Mol Genet Genomics. 2002, 267: 657-6.View ArticleGoogle Scholar
- Singh L, Phillips C, Jones KW: The conserved nucleotide sequences of Bkm, which define sxr in the mouse, are transcribed. Cell. 1984, 35: 111-120.View ArticleGoogle Scholar
- Le MH, Duricka D, Karpen GH: Islands of complex DNA are widespread in Drosophila centric heterochromatin. Genetics. 1995, 141: 283-303.PubMed CentralPubMedGoogle Scholar
- Sun X, Le HD, Wahlstrom JM, Karpen GR: Sequence analysis of a functional Drosophila centromere. Genome Research. 2003, 13: 82-194.View ArticleGoogle Scholar
- Bachtrog D, et al: Distribution of dinucleotide microsatellites in the Drosophila melanogaster genome. Mol Biol Evo. 1999, 16: 602-610.View ArticleGoogle Scholar
- Toth G, Gaspari Z, Jurka J: Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res. 2000, 10: 967-981.PubMed CentralPubMedView ArticleGoogle Scholar
- Katti MV, Ranjekar PK, Gupta VS: Differential distribution of Simple Sequence Repeats in eukaryotic genome sequences. Mol Biol Evol. 2001, 18: 1161-1167.PubMedView ArticleGoogle Scholar
- Subramanian S, Mishra RK, Singh L: Genome-wide analysis of microsatellite repeats in humans: their abundance and density in specific genomic regions. Genome Biology. 2003, 4: R13-PubMed CentralPubMedView ArticleGoogle Scholar
- Subirana JA, Messenguer X: The most frequent short sequences in non-coding DNA. Nucleic Acids Research. 2010, 38: 1172-1181.PubMed CentralPubMedView ArticleGoogle Scholar
- Pardue ML, Lowenhaupt K, Rich A, Nordheim A: (dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function. EMBO J. 1987, 6: 1781-1789.PubMed CentralPubMedGoogle Scholar
- Lowenhaupt K, Rich A, Pardue ML: Nonrandom distribution of long mono- and dinucleotide repeats in Drosophila chromosomes: Correlations with dosage compensation, heterochromatin and recombination. Molecular and Cellular Biology. 1989, 9: 1173-1182.PubMed CentralPubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.