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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

Structure of the centromere of the third chromosome of D. melanogaster.(a) 2.5 Mb physical map across the centromere of chromosome 3. The regions containing the 10 bp satellite repeats and the dodeca satellite repeats appear in green and red, respectively. The segmental duplications from the NOR and from region 2L-29C are indicated in purple and blue, respectively. The position and GenBank number of the eight centromeric scaffolds are also indicated. Abbreviations are: B, BamHI; H, BssHII; E, BstEII, R, EcoRI; N, NaeI; Hp, HpaI; P, PmeI, S, SwaI. (b) Extended chromatin fibers from S2 cells were processed for immunofluorescence with an anti-CID antibody followed by FISH with the dodeca satellite oligo probe. A representative image showing CID immunostaining (green) overlapping with dodeca (red) is shown. Of a total of 43 chromatin fibers stained with the anti-CID antibody, 14 showed co-localization with dodeca, a proportion in agreement with the karyotype of the polyploid S2 cells. CID signals do not encompass all dodeca satellite repeats. A minor number of fibers containing dodeca satellites are not stained with the anti-CID antibody. Scale bar is 5 μm.
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f1: Structure of the centromere of the third chromosome of D. melanogaster.(a) 2.5 Mb physical map across the centromere of chromosome 3. The regions containing the 10 bp satellite repeats and the dodeca satellite repeats appear in green and red, respectively. The segmental duplications from the NOR and from region 2L-29C are indicated in purple and blue, respectively. The position and GenBank number of the eight centromeric scaffolds are also indicated. Abbreviations are: B, BamHI; H, BssHII; E, BstEII, R, EcoRI; N, NaeI; Hp, HpaI; P, PmeI, S, SwaI. (b) Extended chromatin fibers from S2 cells were processed for immunofluorescence with an anti-CID antibody followed by FISH with the dodeca satellite oligo probe. A representative image showing CID immunostaining (green) overlapping with dodeca (red) is shown. Of a total of 43 chromatin fibers stained with the anti-CID antibody, 14 showed co-localization with dodeca, a proportion in agreement with the karyotype of the polyploid S2 cells. CID signals do not encompass all dodeca satellite repeats. A minor number of fibers containing dodeca satellites are not stained with the anti-CID antibody. Scale bar is 5 μm.

Mentions: Since a complete physical map across the centromere should extend from chromosome arm 3L to chromosome arm 3R, we set out to isolate bacterial artificial chromosome (BAC) clones that contain dodeca satellite, and to construct a comprehensive map around the dodeca satellite blocks using 20 restriction enzymes that do not occur within the dodeca satellite. Single and double genomic digests were size-fractionated by pulsed-field gel electrophoresis (PFGE) using a “Waltzer” apparatus40, which gives sharp resolution up to 2 Mb (representative digests are shown in Supplementary Fig. S1). In order to obtain dodeca satellite clones, three D. melanogaster BAC libraries were screened: the RPCI-98 library generated by cloning EcoRI-digested genomic DNA and the CHORI-221 and CHORI-223 libraries generated from sheared genomic DNA. BAC end sequencing and fingerprinting data of the stronger clones were used to construct contigs, and five clones were chosen for complete sequencing: CH221-29J09 (containing rDNA intergenic spacer (IGS) related sequences, cIGS), CH211-27P10 (containing Akap200 related sequences) and BACR19P07, BACR16A01 and BACR12I02 that were also positive for the retrotransposon Circe. The presence of Circe sequences in the centromeric region h53 had previously been reported41. By combining data from the sequence of these BACs and the sequence of whole genome shotgun scaffolds containing dodeca satellite with the results of an accurate restriction site mapping of genomic DNA, it has been possible to determine the position and orientation of the first eight scaffolds in the Release 6 assembly of the chromosome arm 3R (Fig. 1a).


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)

Structure of the centromere of the third chromosome of D. melanogaster.(a) 2.5 Mb physical map across the centromere of chromosome 3. The regions containing the 10 bp satellite repeats and the dodeca satellite repeats appear in green and red, respectively. The segmental duplications from the NOR and from region 2L-29C are indicated in purple and blue, respectively. The position and GenBank number of the eight centromeric scaffolds are also indicated. Abbreviations are: B, BamHI; H, BssHII; E, BstEII, R, EcoRI; N, NaeI; Hp, HpaI; P, PmeI, S, SwaI. (b) Extended chromatin fibers from S2 cells were processed for immunofluorescence with an anti-CID antibody followed by FISH with the dodeca satellite oligo probe. A representative image showing CID immunostaining (green) overlapping with dodeca (red) is shown. Of a total of 43 chromatin fibers stained with the anti-CID antibody, 14 showed co-localization with dodeca, a proportion in agreement with the karyotype of the polyploid S2 cells. CID signals do not encompass all dodeca satellite repeats. A minor number of fibers containing dodeca satellites are not stained with the anti-CID antibody. Scale bar is 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4542561&req=5

f1: Structure of the centromere of the third chromosome of D. melanogaster.(a) 2.5 Mb physical map across the centromere of chromosome 3. The regions containing the 10 bp satellite repeats and the dodeca satellite repeats appear in green and red, respectively. The segmental duplications from the NOR and from region 2L-29C are indicated in purple and blue, respectively. The position and GenBank number of the eight centromeric scaffolds are also indicated. Abbreviations are: B, BamHI; H, BssHII; E, BstEII, R, EcoRI; N, NaeI; Hp, HpaI; P, PmeI, S, SwaI. (b) Extended chromatin fibers from S2 cells were processed for immunofluorescence with an anti-CID antibody followed by FISH with the dodeca satellite oligo probe. A representative image showing CID immunostaining (green) overlapping with dodeca (red) is shown. Of a total of 43 chromatin fibers stained with the anti-CID antibody, 14 showed co-localization with dodeca, a proportion in agreement with the karyotype of the polyploid S2 cells. CID signals do not encompass all dodeca satellite repeats. A minor number of fibers containing dodeca satellites are not stained with the anti-CID antibody. Scale bar is 5 μm.
Mentions: Since a complete physical map across the centromere should extend from chromosome arm 3L to chromosome arm 3R, we set out to isolate bacterial artificial chromosome (BAC) clones that contain dodeca satellite, and to construct a comprehensive map around the dodeca satellite blocks using 20 restriction enzymes that do not occur within the dodeca satellite. Single and double genomic digests were size-fractionated by pulsed-field gel electrophoresis (PFGE) using a “Waltzer” apparatus40, which gives sharp resolution up to 2 Mb (representative digests are shown in Supplementary Fig. S1). In order to obtain dodeca satellite clones, three D. melanogaster BAC libraries were screened: the RPCI-98 library generated by cloning EcoRI-digested genomic DNA and the CHORI-221 and CHORI-223 libraries generated from sheared genomic DNA. BAC end sequencing and fingerprinting data of the stronger clones were used to construct contigs, and five clones were chosen for complete sequencing: CH221-29J09 (containing rDNA intergenic spacer (IGS) related sequences, cIGS), CH211-27P10 (containing Akap200 related sequences) and BACR19P07, BACR16A01 and BACR12I02 that were also positive for the retrotransposon Circe. The presence of Circe sequences in the centromeric region h53 had previously been reported41. By combining data from the sequence of these BACs and the sequence of whole genome shotgun scaffolds containing dodeca satellite with the results of an accurate restriction site mapping of genomic DNA, it has been possible to determine the position and orientation of the first eight scaffolds in the Release 6 assembly of the chromosome arm 3R (Fig. 1a).

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