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The replication of plastid minicircles involves rolling circle intermediates.

Leung SK, Wong JT - Nucleic Acids Res. (2009)

Bottom Line: Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes.APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2-3 kb minicircles, implicating they are not large minicircles.Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR, PR China.

ABSTRACT
Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250-500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6-8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2-3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6-8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

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Patterns of minicircle DNA resolved in 2D-gel. Whole-DNA extract from H. triquetra was subjected to 2D-gel as described in the Methods section. The first dimension is represented horizontally (left to right) and the second dimension vertically (top to bottom). Minicircle DNA were detected with 1.1-kb (A), 0.4-kb (B) and 0.6-kb (C) psbA NCR, and 0.7-kb psbA gene fragment (D). Four images were aligned with respect to their DNA migration in both dimensions of electrophoreses. The putative psbA minicircle spots indicated by the arrows were perfectly aligned. The sizes of hybridization signals can be revealed by the size ladder on the top.
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Figure 4: Patterns of minicircle DNA resolved in 2D-gel. Whole-DNA extract from H. triquetra was subjected to 2D-gel as described in the Methods section. The first dimension is represented horizontally (left to right) and the second dimension vertically (top to bottom). Minicircle DNA were detected with 1.1-kb (A), 0.4-kb (B) and 0.6-kb (C) psbA NCR, and 0.7-kb psbA gene fragment (D). Four images were aligned with respect to their DNA migration in both dimensions of electrophoreses. The putative psbA minicircle spots indicated by the arrows were perfectly aligned. The sizes of hybridization signals can be revealed by the size ladder on the top.

Mentions: Heterocapsa triquetra total DNA was resolved in 2D-gel and detected with the psbA minicircle fragments (Figure 4A). The patterns of the replicative arcs resembled a diagonal with a slightly curved shape extending beyond 6 kb, as well as several strong signal spots. These spots were distributed across the diagonal. The full length 1.1 kb NCR probed more species of minicircular DNA, while the 0.4 kb and 0.6 kb NCR fragments detected less. The psbA gene fragment that specifically hybridize to the psbA minicircle showed one strong spot and two weak spots, resembling the observation in PFGE, as well as a very weak thread of signals joining these spots. Short curved diagonals up to 3 kb were also observed underneath the long diagonals, which contained spots of sizes similar to those in the long diagonals, but with slightly lower signal intensities. By comparing the four patterns, strong spots of minicircular DNA were observed at ∼1.6 kb, ∼2 kb, ∼2.5 kb and ∼3 kb, weaker spots were observed at ∼4 kb, ∼4.5 kb and ∼5.2 kb (Figure 4B).Figure 4.


The replication of plastid minicircles involves rolling circle intermediates.

Leung SK, Wong JT - Nucleic Acids Res. (2009)

Patterns of minicircle DNA resolved in 2D-gel. Whole-DNA extract from H. triquetra was subjected to 2D-gel as described in the Methods section. The first dimension is represented horizontally (left to right) and the second dimension vertically (top to bottom). Minicircle DNA were detected with 1.1-kb (A), 0.4-kb (B) and 0.6-kb (C) psbA NCR, and 0.7-kb psbA gene fragment (D). Four images were aligned with respect to their DNA migration in both dimensions of electrophoreses. The putative psbA minicircle spots indicated by the arrows were perfectly aligned. The sizes of hybridization signals can be revealed by the size ladder on the top.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665238&req=5

Figure 4: Patterns of minicircle DNA resolved in 2D-gel. Whole-DNA extract from H. triquetra was subjected to 2D-gel as described in the Methods section. The first dimension is represented horizontally (left to right) and the second dimension vertically (top to bottom). Minicircle DNA were detected with 1.1-kb (A), 0.4-kb (B) and 0.6-kb (C) psbA NCR, and 0.7-kb psbA gene fragment (D). Four images were aligned with respect to their DNA migration in both dimensions of electrophoreses. The putative psbA minicircle spots indicated by the arrows were perfectly aligned. The sizes of hybridization signals can be revealed by the size ladder on the top.
Mentions: Heterocapsa triquetra total DNA was resolved in 2D-gel and detected with the psbA minicircle fragments (Figure 4A). The patterns of the replicative arcs resembled a diagonal with a slightly curved shape extending beyond 6 kb, as well as several strong signal spots. These spots were distributed across the diagonal. The full length 1.1 kb NCR probed more species of minicircular DNA, while the 0.4 kb and 0.6 kb NCR fragments detected less. The psbA gene fragment that specifically hybridize to the psbA minicircle showed one strong spot and two weak spots, resembling the observation in PFGE, as well as a very weak thread of signals joining these spots. Short curved diagonals up to 3 kb were also observed underneath the long diagonals, which contained spots of sizes similar to those in the long diagonals, but with slightly lower signal intensities. By comparing the four patterns, strong spots of minicircular DNA were observed at ∼1.6 kb, ∼2 kb, ∼2.5 kb and ∼3 kb, weaker spots were observed at ∼4 kb, ∼4.5 kb and ∼5.2 kb (Figure 4B).Figure 4.

Bottom Line: Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes.APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2-3 kb minicircles, implicating they are not large minicircles.Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR, PR China.

ABSTRACT
Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250-500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6-8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2-3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6-8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

Show MeSH
Related in: MedlinePlus