Abstract
BackgroundIn 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.ResultsThis 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.ConclusionThese 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.
Highlights
In recent years, substantial progress has been made in understanding the organization of sequences in heterochromatin regions containing single-copy genes and transposable elements
Chromosomal localization of pentanucleotide DNA satellites by Non-denaturing FISH (ND-FISH) To verify whether ND-FISH was a suitable technique for identifying clusters of simple sequence repeats (SSRs) in the heterochromatin of D. melanogaster, the chromosomal distributions of five pentanucleotide probes were analyzed: (AAGAG)3, (AAGAC)3, (AATAT)3, (AATAC)3 and (AACAC)3 (Figure 1)
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)
Summary
Substantial progress has been made in understanding the organization of sequences in heterochromatin regions containing single-copy genes and transposable elements. The sequence and organization of tandem repeat DNA sequences, which are by far the majority fraction of D. melanogaster heterochromatin, are little understood. One of the most enigmatic aspects of genome organization in multicellular eukaryotes is the regionalization of chromosomes into euchromatin and heterochromatin domains. 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. Polytene chromosomes, which have proved useful in mapping euchromatin regions, provide minimal resolution in heterochromatin analyses. Mitotic rather than polytene chromosomes are preferable for chromosome mapping of heterochromatic sequences [7]
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