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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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Detection of eccDNA molecules derived from Vicia faba FokI repeats in different tissues and under stress conditions. (A) Schematic outline of the migration patterns of linear and circular DNA forms on 2-D gel electrophoresis. (B-F) Detection of eccDNA in genomic DNA samples of V. faba separated on 2-D electrophoresis (0.4% and 1% agarose), Southern blotted and hybridized with FokI-derived probe. The DNA was isolated from root meristems (B), young (C) or old leaves (D), and from wounded leaves one (E) and two (F) days after the treatment.
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Figure 1: Detection of eccDNA molecules derived from Vicia faba FokI repeats in different tissues and under stress conditions. (A) Schematic outline of the migration patterns of linear and circular DNA forms on 2-D gel electrophoresis. (B-F) Detection of eccDNA in genomic DNA samples of V. faba separated on 2-D electrophoresis (0.4% and 1% agarose), Southern blotted and hybridized with FokI-derived probe. The DNA was isolated from root meristems (B), young (C) or old leaves (D), and from wounded leaves one (E) and two (F) days after the treatment.

Mentions: We employed two-dimensional (2-D) agarose gel electrophoresis [22] followed by Southern blot hybridization to examine the presence of eccDNA in extracts of total genomic DNA from selected plant species. In addition to separating DNA molecules based on their size, 2-D electrophoresis also allows resolution based on their structure, resulting in formation of separated arcs on the gel representing linear and various conformations of circular DNA molecules (Fig. 1A). In our initial experiments we performed 2-D electrophoresis in 0.4% agarose in the first dimension, and in 1% agarose supplemented with ethidium bromide in the second dimension, which provided efficient resolution of linear and circular molecules from over 9 kb down to 3 kb as assessed from migration of linear and supercoiled or open circular marker molecules (not shown). These conditions were then used to analyze DNA samples of Vicia faba, a species containing highly abundant satellite repeat FokI [23,24] (Table 1). A strong arc of linear DNA and a weaker one corresponding to open circular molecules were revealed after hybridization with FokI probe. We used this species further for investigating FokI eccDNA levels in different tissues including young/developing or old leaves and root meristems (Fig. 1B–D), and also in leaf tissues stressed by wounding (Fig. 1E–F). However, we did not observe significant differences in eccDNA signals between these samples, suggesting that there are comparable levels of circular molecules derived from FokI satellite in all investigated V. faba tissues and that these levels are not significantly affected by stress conditions. As only open circular molecules were detected and there was no hybridization signal corresponding to covalently closed circular DNA, we tested whether this could be a result of damaging closed circles during DNA isolation. However, including supercoiled control plasmid DNA into tissue samples at the beginning of the procedure revealed that the isolation does not lead to significant nicking or other degradation of this DNA, and thus that open circles are the predominant form of satellite eccDNA in the examined tissues.


Survey of extrachromosomal circular DNA derived from plant satellite repeats.

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

Detection of eccDNA molecules derived from Vicia faba FokI repeats in different tissues and under stress conditions. (A) Schematic outline of the migration patterns of linear and circular DNA forms on 2-D gel electrophoresis. (B-F) Detection of eccDNA in genomic DNA samples of V. faba separated on 2-D electrophoresis (0.4% and 1% agarose), Southern blotted and hybridized with FokI-derived probe. The DNA was isolated from root meristems (B), young (C) or old leaves (D), and from wounded leaves one (E) and two (F) days after the treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543021&req=5

Figure 1: Detection of eccDNA molecules derived from Vicia faba FokI repeats in different tissues and under stress conditions. (A) Schematic outline of the migration patterns of linear and circular DNA forms on 2-D gel electrophoresis. (B-F) Detection of eccDNA in genomic DNA samples of V. faba separated on 2-D electrophoresis (0.4% and 1% agarose), Southern blotted and hybridized with FokI-derived probe. The DNA was isolated from root meristems (B), young (C) or old leaves (D), and from wounded leaves one (E) and two (F) days after the treatment.
Mentions: We employed two-dimensional (2-D) agarose gel electrophoresis [22] followed by Southern blot hybridization to examine the presence of eccDNA in extracts of total genomic DNA from selected plant species. In addition to separating DNA molecules based on their size, 2-D electrophoresis also allows resolution based on their structure, resulting in formation of separated arcs on the gel representing linear and various conformations of circular DNA molecules (Fig. 1A). In our initial experiments we performed 2-D electrophoresis in 0.4% agarose in the first dimension, and in 1% agarose supplemented with ethidium bromide in the second dimension, which provided efficient resolution of linear and circular molecules from over 9 kb down to 3 kb as assessed from migration of linear and supercoiled or open circular marker molecules (not shown). These conditions were then used to analyze DNA samples of Vicia faba, a species containing highly abundant satellite repeat FokI [23,24] (Table 1). A strong arc of linear DNA and a weaker one corresponding to open circular molecules were revealed after hybridization with FokI probe. We used this species further for investigating FokI eccDNA levels in different tissues including young/developing or old leaves and root meristems (Fig. 1B–D), and also in leaf tissues stressed by wounding (Fig. 1E–F). However, we did not observe significant differences in eccDNA signals between these samples, suggesting that there are comparable levels of circular molecules derived from FokI satellite in all investigated V. faba tissues and that these levels are not significantly affected by stress conditions. As only open circular molecules were detected and there was no hybridization signal corresponding to covalently closed circular DNA, we tested whether this could be a result of damaging closed circles during DNA isolation. However, including supercoiled control plasmid DNA into tissue samples at the beginning of the procedure revealed that the isolation does not lead to significant nicking or other degradation of this DNA, and thus that open circles are the predominant form of satellite eccDNA in the examined tissues.

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