Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Photomicrographs showing the motif-dependent chromosomal distribution of the pentanucleotide probes in the salivary gland (a, c, f, g, i, m and n) and neuroblast cells (a, b, d, e, h, j, k, l, m and o) of male and female D. melanogaster after ND-FISH and DAPI staining. Each panel shows individual and/or merged images to facilitate the visualization of the signals (green or red for digoxigenin- and biotin-labelled probes respectively) with respect to the DAPI (blue) banding pattern. In some samples interphase and mitotic neuroblast nuclei are shown in the same panels; both polytene and diploid nuclei are shown in a and m. Mitotic chromosomes are identified as well as polytene regions of interest. An example of two-colour ND-FISH is shown in e. Note that only the diffuse chromocentre (CR) observed in polytene nuclei is enriched in pentanucleotide SSRs, which localize to specific heterochromatic regions in mitotic chromosomes as shown in Figure 4. The arrows point to low intensity signals. Scale bar: 5 μm, except in a and m, in which it represents 25 μm.

Figure 2

Chromosomal distribution of the mono- and dinucleotide SSRs in the salivary gland (a, d, e, and h) and neuroblast cells (b, c, f, i and j) of D. melanogaster after ND-FISH and DAPI staining. Both polytene chromosomes and diploid nuclei are shown in g; examples of two-colour ND-FISH with (AAGAG)₅ are shown in h (insets) and j. Each panel shows individual and/or merged images to facilitate the appreciation of the distribution of signals (green or red from digoxigenin- and biotin-labelled probes respectively) and identification of chromosomes. The localization of the signals with respect to the heterochromatic DAPI banding pattern in mitotic chromosomes is shown in Figure 4. Note the high concentration of A, C, AC and AG SSRs on polytene X chromosomes; this contrasts with the lesser presence of these repeats on chromosome 4. An enlarged view of the A repeat signals at the base of chromosome X (square) and chromosome 4 is shown at the right of panel a. The arrows point chromosomes 4; CR, chromocentre. Scale bar = 5 μm and 25 μm in (pro)metaphase and polytene nuclei respectively.

Figure 3

The non-random chromosomal distribution of several tri- and tetranucleotide SSRs in polytene (panels a, d, e, g, h, i, j, l and o; scale bar 25 μm) and mitotic (b, c, f, k, m, n, p and q; scale bar 5 μm) nuclei of D. melanogaster, as shown by ND-FISH with the indicated biotinylated probes. Each panel shows individual and/or merged images to facilitate the visualization of the red signals with respect to the DAPI (blue) staining and to allow chromosome identification. The view of the localization of the AAT repeats in the base of chromosome X is enlarged in h. A detailed view of the chromocentre (CR) after ND-FISH with (ACT)₅, (GACA)₄ and (GATA)₄ is shown in e and in the inserts in panels l and o respectively. The arrows point to chromosomes X and 4 in b and k respectively.

Figure 4

Distribution of several SSRs in the D. melanogaster cytogenetic reference map of the heterochromatic regions (h1 to h61) derived from Pimpinelli et al. [26] (the darker the block, the brighter the DAPI intensity; centromeres [C] and rDNA are also indicated). At the resolution in Figure 1, the distribution of the pentanucleotide repeats is the same as reported by Lohe et al. [10] and Mukani et al. [11]. Thus, the localizations of these pentanucleotide SSRs are indicated below the chromosomes as previously mapped. Additional localizations to those reported are shown by arrows. Lines above chromosomes represent the novel SSRs described in the present work (Figs. 2 and 3). Note that some sequence has been mapped to chromosome regions defined by several heterochromatic bands, but neither the physical size nor intensity of the fluorescence signals is represented here.

Figure 5

Organization of representative SSRs in extended DNA fibres from neuroblast nuclei of D. melanogaster after ND-FISH with the indicated probes (green or red signals from digoxigenin- and biotin-labelled probes respectively) and DAPI (blue) staining. An example of two colour fibre ND-FISH in haloed nuclei with (AAGAC)₃ (red) and (AACAC)₃ (green) is shown in a. Examples of fluorescent signal patterns in fibres stretched to different degrees are shown in e (square insets). Note the characteristic beaded nature of the signals in the highly extended fibres. A representative of the gaps observed between the continuous track of beaded signals is amplified in f. Scale bar = 10 μm, except in f in which it represents 5 μm. Every micrometer represents 3 kb of the highly stretched DNA.

Figure 6

Southern hybridization patterns of genomic DNA from female and male D. melanogaster digested by AluI, HinfI and RsaI and fractionated by conventional 1% agarose gel electrophoresis (A). Three filter transfers were sequentially hybridized with the indicated digoxigenin end-labelled SSR probes (B to K). Size markers (M) are given in kb.