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Survey of extrachromosomal circular DNA derived from plant satellite repeats.

Navrátilová A, Koblízková A, Macas J - BMC Plant Biol. (2008)

Bottom Line: EccDNA occurred in the form of nicked circles ranging from hundreds to over eight thousand nucleotides in size.Within this range the circular molecules occurred preferentially in discrete size intervals corresponding to multiples of monomer or higher-order repeat lengths.This work demonstrated that satellite repeat-derived eccDNA is common in plant genomes and thus it can be seriously considered as a potential intermediate in processes driving satellite repeat evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Centre ASCR, Institute of Plant Molecular Biology, Branisovská 31, Ceské Budejovice, CZ-37005, Czech Republic. navratil@umbr.cas.cz

ABSTRACT

Background: Satellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes.

Results: We performed a survey of eccDNA corresponding to nine different families and three subfamilies of satellite repeats in ten species from various genera of higher plants (Arabidopsis, Oryza, Pisum, Secale, Triticum and Vicia). The repeats selected for this study differed in their monomer length, abundance, and chromosomal localization in individual species. Using two-dimensional agarose gel electrophoresis followed by Southern blotting, eccDNA molecules corresponding to all examined satellites were detected. EccDNA occurred in the form of nicked circles ranging from hundreds to over eight thousand nucleotides in size. Within this range the circular molecules occurred preferentially in discrete size intervals corresponding to multiples of monomer or higher-order repeat lengths.

Conclusion: This work demonstrated that satellite repeat-derived eccDNA is common in plant genomes and thus it can be seriously considered as a potential intermediate in processes driving satellite repeat evolution. The observed size distribution of circular molecules suggests that they are most likely generated by molecular mechanisms based on homologous recombination requiring long stretches of sequence similarity.

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Survey of eccDNA derived from various plant satellites. Samples of genomic DNA enriched for circular DNA were separated on 0.7% and 2% 2-D gels, blotted and hybridized with satellite-specific probes. The arcs corresponding to linear (L) and open circle (OC) DNA are indicated with black arrowheads and positions of the longest (1,674 bp) and the shortest (558 bp) OC markers are shown as blue spots. The extra arc of unknown origin is indicated with black arrow on panels A, B, D and E. (A-B) Detection of Sc119 repeats in Secale cereale, demonstrating the effect of single-stranded DNA degradation by mung bean nuclease (B) compared to untreated control (A). Figure insets contain magnified regions marked by the rectangles showing the effect of the nuclease treatment on shape of the spots. All other samples shown on panels C-L were also treated with mung bean nuclease. (C) Afa repeats in Triticum aestivum, (D) IGS-like in Vicia sativa, (E) CentO in Oryza sativa, (F) FokI in V. faba, (G) PisTR-B in Pisum sativum (this sample was resolved on 1.5% and 2% 2-D gels), (H) VicTR-B in V. grandiflora, (I) VG-V subfamily of VicTR-B in V. grandiflora, (J) VicTR-A in V. pannonica detected using PCR probe including a mixture of genomic VicTR-A sequences, (K-L) VicTR-A in V. pannonica detected using specific clones c653 (K) and c666 (L).
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Figure 3: Survey of eccDNA derived from various plant satellites. Samples of genomic DNA enriched for circular DNA were separated on 0.7% and 2% 2-D gels, blotted and hybridized with satellite-specific probes. The arcs corresponding to linear (L) and open circle (OC) DNA are indicated with black arrowheads and positions of the longest (1,674 bp) and the shortest (558 bp) OC markers are shown as blue spots. The extra arc of unknown origin is indicated with black arrow on panels A, B, D and E. (A-B) Detection of Sc119 repeats in Secale cereale, demonstrating the effect of single-stranded DNA degradation by mung bean nuclease (B) compared to untreated control (A). Figure insets contain magnified regions marked by the rectangles showing the effect of the nuclease treatment on shape of the spots. All other samples shown on panels C-L were also treated with mung bean nuclease. (C) Afa repeats in Triticum aestivum, (D) IGS-like in Vicia sativa, (E) CentO in Oryza sativa, (F) FokI in V. faba, (G) PisTR-B in Pisum sativum (this sample was resolved on 1.5% and 2% 2-D gels), (H) VicTR-B in V. grandiflora, (I) VG-V subfamily of VicTR-B in V. grandiflora, (J) VicTR-A in V. pannonica detected using PCR probe including a mixture of genomic VicTR-A sequences, (K-L) VicTR-A in V. pannonica detected using specific clones c653 (K) and c666 (L).

Mentions: Extrachromosomal circular molecules were detected for all nine satellite families and three subfamilies tested, and in all cases they occurred as open circles (Fig. 3). The size of the eccDNA molecules ranged from over 8 kb down to 500 bp and in most cases their hybridization signals formed discrete spots corresponding to multiples of monomer or higher-order unit lengths (Table 1). However, there was often a continuous smear underlying these spots, as shown on Fig. 3A for Sc119 satellite of S. cereale. Treating the samples with mung bean nuclease, which specifically degrades single-stranded but not double-stranded or nicked DNA almost completely removed the smear, resulting in more round and discrete spots (Fig. 3B), thus suggesting that the smear was composed of partially single-stranded circular molecules. In addition to the arcs representing circular and linear DNA molecules, in some cases we encountered hybridization signals forming a relatively fuzzy arc that could not be assigned to linear, covalently closed supercoiled or open circular molecules by co-migration with the respective markers. The signal was reproducibly detected for satellites from Secale cereale, Vicia sativa and Oryza sativa (Fig. 3B, D, E). As it was present even in mung bean nuclease-treated samples, its composition from single-stranded DNA or RNA could be ruled out. Thus, we tested whether it could be affected by treatment with RNaseH or RNaseH followed by mung bean nuclease. However, these treatments had no effect, implying that the extra arc did not include hybrid DNA:RNA molecules (data not shown).


Survey of extrachromosomal circular DNA derived from plant satellite repeats.

Navrátilová A, Koblízková A, Macas J - BMC Plant Biol. (2008)

Survey of eccDNA derived from various plant satellites. Samples of genomic DNA enriched for circular DNA were separated on 0.7% and 2% 2-D gels, blotted and hybridized with satellite-specific probes. The arcs corresponding to linear (L) and open circle (OC) DNA are indicated with black arrowheads and positions of the longest (1,674 bp) and the shortest (558 bp) OC markers are shown as blue spots. The extra arc of unknown origin is indicated with black arrow on panels A, B, D and E. (A-B) Detection of Sc119 repeats in Secale cereale, demonstrating the effect of single-stranded DNA degradation by mung bean nuclease (B) compared to untreated control (A). Figure insets contain magnified regions marked by the rectangles showing the effect of the nuclease treatment on shape of the spots. All other samples shown on panels C-L were also treated with mung bean nuclease. (C) Afa repeats in Triticum aestivum, (D) IGS-like in Vicia sativa, (E) CentO in Oryza sativa, (F) FokI in V. faba, (G) PisTR-B in Pisum sativum (this sample was resolved on 1.5% and 2% 2-D gels), (H) VicTR-B in V. grandiflora, (I) VG-V subfamily of VicTR-B in V. grandiflora, (J) VicTR-A in V. pannonica detected using PCR probe including a mixture of genomic VicTR-A sequences, (K-L) VicTR-A in V. pannonica detected using specific clones c653 (K) and c666 (L).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Survey of eccDNA derived from various plant satellites. Samples of genomic DNA enriched for circular DNA were separated on 0.7% and 2% 2-D gels, blotted and hybridized with satellite-specific probes. The arcs corresponding to linear (L) and open circle (OC) DNA are indicated with black arrowheads and positions of the longest (1,674 bp) and the shortest (558 bp) OC markers are shown as blue spots. The extra arc of unknown origin is indicated with black arrow on panels A, B, D and E. (A-B) Detection of Sc119 repeats in Secale cereale, demonstrating the effect of single-stranded DNA degradation by mung bean nuclease (B) compared to untreated control (A). Figure insets contain magnified regions marked by the rectangles showing the effect of the nuclease treatment on shape of the spots. All other samples shown on panels C-L were also treated with mung bean nuclease. (C) Afa repeats in Triticum aestivum, (D) IGS-like in Vicia sativa, (E) CentO in Oryza sativa, (F) FokI in V. faba, (G) PisTR-B in Pisum sativum (this sample was resolved on 1.5% and 2% 2-D gels), (H) VicTR-B in V. grandiflora, (I) VG-V subfamily of VicTR-B in V. grandiflora, (J) VicTR-A in V. pannonica detected using PCR probe including a mixture of genomic VicTR-A sequences, (K-L) VicTR-A in V. pannonica detected using specific clones c653 (K) and c666 (L).
Mentions: Extrachromosomal circular molecules were detected for all nine satellite families and three subfamilies tested, and in all cases they occurred as open circles (Fig. 3). The size of the eccDNA molecules ranged from over 8 kb down to 500 bp and in most cases their hybridization signals formed discrete spots corresponding to multiples of monomer or higher-order unit lengths (Table 1). However, there was often a continuous smear underlying these spots, as shown on Fig. 3A for Sc119 satellite of S. cereale. Treating the samples with mung bean nuclease, which specifically degrades single-stranded but not double-stranded or nicked DNA almost completely removed the smear, resulting in more round and discrete spots (Fig. 3B), thus suggesting that the smear was composed of partially single-stranded circular molecules. In addition to the arcs representing circular and linear DNA molecules, in some cases we encountered hybridization signals forming a relatively fuzzy arc that could not be assigned to linear, covalently closed supercoiled or open circular molecules by co-migration with the respective markers. The signal was reproducibly detected for satellites from Secale cereale, Vicia sativa and Oryza sativa (Fig. 3B, D, E). As it was present even in mung bean nuclease-treated samples, its composition from single-stranded DNA or RNA could be ruled out. Thus, we tested whether it could be affected by treatment with RNaseH or RNaseH followed by mung bean nuclease. However, these treatments had no effect, implying that the extra arc did not include hybrid DNA:RNA molecules (data not shown).

Bottom Line: EccDNA occurred in the form of nicked circles ranging from hundreds to over eight thousand nucleotides in size.Within this range the circular molecules occurred preferentially in discrete size intervals corresponding to multiples of monomer or higher-order repeat lengths.This work demonstrated that satellite repeat-derived eccDNA is common in plant genomes and thus it can be seriously considered as a potential intermediate in processes driving satellite repeat evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Centre ASCR, Institute of Plant Molecular Biology, Branisovská 31, Ceské Budejovice, CZ-37005, Czech Republic. navratil@umbr.cas.cz

ABSTRACT

Background: Satellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes.

Results: We performed a survey of eccDNA corresponding to nine different families and three subfamilies of satellite repeats in ten species from various genera of higher plants (Arabidopsis, Oryza, Pisum, Secale, Triticum and Vicia). The repeats selected for this study differed in their monomer length, abundance, and chromosomal localization in individual species. Using two-dimensional agarose gel electrophoresis followed by Southern blotting, eccDNA molecules corresponding to all examined satellites were detected. EccDNA occurred in the form of nicked circles ranging from hundreds to over eight thousand nucleotides in size. Within this range the circular molecules occurred preferentially in discrete size intervals corresponding to multiples of monomer or higher-order repeat lengths.

Conclusion: This work demonstrated that satellite repeat-derived eccDNA is common in plant genomes and thus it can be seriously considered as a potential intermediate in processes driving satellite repeat evolution. The observed size distribution of circular molecules suggests that they are most likely generated by molecular mechanisms based on homologous recombination requiring long stretches of sequence similarity.

Show MeSH
Related in: MedlinePlus