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The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs.

Garavís M, Méndez-Lago M, Gabelica V, Whitehead SL, González C, Villasante A - Sci Rep (2015)

Bottom Line: In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements).Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs.The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049 Madrid, Spain.

ABSTRACT
Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

No MeSH data available.


Related in: MedlinePlus

The 10 bp satellite DNA localizes on the third chromosome at h52p instead of h48.(a) Metaphase chromosomes counterstained with DAPI. (b) Hybridization signals from a dodeca satellite probe (in red). (c) Hybridization signals from a 10 bp satellite probe (in green). (d) Hybridization signals superimposed with DAPI-stained chromosomes. The Scale bar is 2 μm. (e) Diagram representing the heterochromatic regions39 of chromosomes 2 (regions 35–46) and 3 (regions 47–58) showing the localization of the 10 bp (in green) and dodeca (in red) satellites. The position of the centromeres (C) is indicated. (f) High molecular weight DNA from red e embryos was digested with BssHII, electrophoresed through a 1% (w/v) agarose gel in a “Waltzer” apparatus at 150 V for 24 h with a 130 s pulse time, blotted onto a nylon filter and hybridized successively with the dodeca satellite probe pBK6E218 at 68 °C and with the 10 bp satellite probe 5′-AATAACATAGAATAACATAGAATAACATAGAATAACATAGAATAACATAG-3′ at 50 °C. The asterisks indicate a 1.2 Mb fragment that hybridizes with both probes. (g) DNA sequence showing a junction between 10 bp satellite repeats (in green) and dodeca satellite repeats (in red).
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f2: The 10 bp satellite DNA localizes on the third chromosome at h52p instead of h48.(a) Metaphase chromosomes counterstained with DAPI. (b) Hybridization signals from a dodeca satellite probe (in red). (c) Hybridization signals from a 10 bp satellite probe (in green). (d) Hybridization signals superimposed with DAPI-stained chromosomes. The Scale bar is 2 μm. (e) Diagram representing the heterochromatic regions39 of chromosomes 2 (regions 35–46) and 3 (regions 47–58) showing the localization of the 10 bp (in green) and dodeca (in red) satellites. The position of the centromeres (C) is indicated. (f) High molecular weight DNA from red e embryos was digested with BssHII, electrophoresed through a 1% (w/v) agarose gel in a “Waltzer” apparatus at 150 V for 24 h with a 130 s pulse time, blotted onto a nylon filter and hybridized successively with the dodeca satellite probe pBK6E218 at 68 °C and with the 10 bp satellite probe 5′-AATAACATAGAATAACATAGAATAACATAGAATAACATAGAATAACATAG-3′ at 50 °C. The asterisks indicate a 1.2 Mb fragment that hybridizes with both probes. (g) DNA sequence showing a junction between 10 bp satellite repeats (in green) and dodeca satellite repeats (in red).

Mentions: During our effort to identify the putative block of simple sequence DNA in the flanking region of the dodeca satellite block 1, we repeated the cytological mapping of the 10 bp satellite (AATAACATAG)n using a fluorescent probe, which improves sensitivity and resolution with respect to results obtained with tritiated probes22. The 10 bp satellite had been mapped by22 to region h37 on the second chromosome (contiguous to the centromeric region h38) and to region h48 on the third chromosome (far away from the centromeric region h53). Unexpectedly, FISH experiments with dodeca and 10 bp satellite probes revealed no additional sites for the 10 bp satellite, but showed a change in its location on the third chromosome from h48 to h52p, a position which is very close to dodeca satellite (Fig. 2a–e). To investigate further the possibility that the flanking satellite DNA corresponds to the 10 bp satellite, we asked whether the 1.2 Mb BssHII fragment containing both dodeca satellite and flanking satellite sequences would hybridize with the 10 bp satellite probe. To this end, genomic DNA was digested with BssHII, size-fractionated by PFGE, transferred to a nylon membrane, hybridized with the dodeca satellite probe and then stripped and re-hybridized with the 10 bp satellite probe. As can be seen in Fig. 2f, there is a 1.2 Mb fragment (labeled with an asterisk) that hybridizes with both probes. Finally, the junction between 10 bp satellite and dodeca satellite sequences was found by searching the Trace Archive database (Fig. 2g). This result indicates that the 10 bp satellite DNA is physically linked to the dodeca satellite DNA. Here, it is important to remember that PROD, a protein required for centromere condensation45 and that specifically recognizes the 10 bp satellite45, is located near but not in the CID-containing chromatin46. Therefore, the physical map constructed comprises two adjacent chromatin domains with distinct functions. Although we have not completed the assembly at a base pair level, the sequence obtained from the five newly sequenced BAC clones, together with pre-existing contigs, with the identification of large blocks of several DNA satellites, and with the correct re-location of the 10 bp satellite from h48 to h52p, represent the most comprehensive physical map and assembly across the centromere of chromosome 3 of Drosophila melanogaster.


The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs.

Garavís M, Méndez-Lago M, Gabelica V, Whitehead SL, González C, Villasante A - Sci Rep (2015)

The 10 bp satellite DNA localizes on the third chromosome at h52p instead of h48.(a) Metaphase chromosomes counterstained with DAPI. (b) Hybridization signals from a dodeca satellite probe (in red). (c) Hybridization signals from a 10 bp satellite probe (in green). (d) Hybridization signals superimposed with DAPI-stained chromosomes. The Scale bar is 2 μm. (e) Diagram representing the heterochromatic regions39 of chromosomes 2 (regions 35–46) and 3 (regions 47–58) showing the localization of the 10 bp (in green) and dodeca (in red) satellites. The position of the centromeres (C) is indicated. (f) High molecular weight DNA from red e embryos was digested with BssHII, electrophoresed through a 1% (w/v) agarose gel in a “Waltzer” apparatus at 150 V for 24 h with a 130 s pulse time, blotted onto a nylon filter and hybridized successively with the dodeca satellite probe pBK6E218 at 68 °C and with the 10 bp satellite probe 5′-AATAACATAGAATAACATAGAATAACATAGAATAACATAGAATAACATAG-3′ at 50 °C. The asterisks indicate a 1.2 Mb fragment that hybridizes with both probes. (g) DNA sequence showing a junction between 10 bp satellite repeats (in green) and dodeca satellite repeats (in red).
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Related In: Results  -  Collection

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Show All Figures
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f2: The 10 bp satellite DNA localizes on the third chromosome at h52p instead of h48.(a) Metaphase chromosomes counterstained with DAPI. (b) Hybridization signals from a dodeca satellite probe (in red). (c) Hybridization signals from a 10 bp satellite probe (in green). (d) Hybridization signals superimposed with DAPI-stained chromosomes. The Scale bar is 2 μm. (e) Diagram representing the heterochromatic regions39 of chromosomes 2 (regions 35–46) and 3 (regions 47–58) showing the localization of the 10 bp (in green) and dodeca (in red) satellites. The position of the centromeres (C) is indicated. (f) High molecular weight DNA from red e embryos was digested with BssHII, electrophoresed through a 1% (w/v) agarose gel in a “Waltzer” apparatus at 150 V for 24 h with a 130 s pulse time, blotted onto a nylon filter and hybridized successively with the dodeca satellite probe pBK6E218 at 68 °C and with the 10 bp satellite probe 5′-AATAACATAGAATAACATAGAATAACATAGAATAACATAGAATAACATAG-3′ at 50 °C. The asterisks indicate a 1.2 Mb fragment that hybridizes with both probes. (g) DNA sequence showing a junction between 10 bp satellite repeats (in green) and dodeca satellite repeats (in red).
Mentions: During our effort to identify the putative block of simple sequence DNA in the flanking region of the dodeca satellite block 1, we repeated the cytological mapping of the 10 bp satellite (AATAACATAG)n using a fluorescent probe, which improves sensitivity and resolution with respect to results obtained with tritiated probes22. The 10 bp satellite had been mapped by22 to region h37 on the second chromosome (contiguous to the centromeric region h38) and to region h48 on the third chromosome (far away from the centromeric region h53). Unexpectedly, FISH experiments with dodeca and 10 bp satellite probes revealed no additional sites for the 10 bp satellite, but showed a change in its location on the third chromosome from h48 to h52p, a position which is very close to dodeca satellite (Fig. 2a–e). To investigate further the possibility that the flanking satellite DNA corresponds to the 10 bp satellite, we asked whether the 1.2 Mb BssHII fragment containing both dodeca satellite and flanking satellite sequences would hybridize with the 10 bp satellite probe. To this end, genomic DNA was digested with BssHII, size-fractionated by PFGE, transferred to a nylon membrane, hybridized with the dodeca satellite probe and then stripped and re-hybridized with the 10 bp satellite probe. As can be seen in Fig. 2f, there is a 1.2 Mb fragment (labeled with an asterisk) that hybridizes with both probes. Finally, the junction between 10 bp satellite and dodeca satellite sequences was found by searching the Trace Archive database (Fig. 2g). This result indicates that the 10 bp satellite DNA is physically linked to the dodeca satellite DNA. Here, it is important to remember that PROD, a protein required for centromere condensation45 and that specifically recognizes the 10 bp satellite45, is located near but not in the CID-containing chromatin46. Therefore, the physical map constructed comprises two adjacent chromatin domains with distinct functions. Although we have not completed the assembly at a base pair level, the sequence obtained from the five newly sequenced BAC clones, together with pre-existing contigs, with the identification of large blocks of several DNA satellites, and with the correct re-location of the 10 bp satellite from h48 to h52p, represent the most comprehensive physical map and assembly across the centromere of chromosome 3 of Drosophila melanogaster.

Bottom Line: In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements).Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs.The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049 Madrid, Spain.

ABSTRACT
Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

No MeSH data available.


Related in: MedlinePlus