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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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AFM images of minicircular DNA. Minicircular DNA of 6–8 kb fractions (A–G) and ∼3 kb fraction (H–J) were subjected to AFM. Images (A, E and G) were performed by scanning probe microscope from Veeco, while the rest (B–D, F and H–J) were by NanoScope III from Digital Instruments. The ethidium bromide stained gel on the left indicates the regions for DNA extractions (gDNA = genomic DNA of H. triquetra). The heights of objects in the AFM images can be denoted by the Z-scale on the right-handed top corner.
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Figure 7: AFM images of minicircular DNA. Minicircular DNA of 6–8 kb fractions (A–G) and ∼3 kb fraction (H–J) were subjected to AFM. Images (A, E and G) were performed by scanning probe microscope from Veeco, while the rest (B–D, F and H–J) were by NanoScope III from Digital Instruments. The ethidium bromide stained gel on the left indicates the regions for DNA extractions (gDNA = genomic DNA of H. triquetra). The heights of objects in the AFM images can be denoted by the Z-scale on the right-handed top corner.

Mentions: AFM is a useful tool to study topology of objects at the nanoscale. It has been used in biological research to image DNA and protein/DNA complexes (37,38). To directly investigate the shape of those double-sized minicircular DNA, AFM of the 6–8 kb fractions was performed. Minicircular DNA of the APBs as detected by Southern blot analysis was adsorbed on smooth surface mica and imaged with AFM. Figure 7 illustrates a number of images of thread-like structures, putative DNA species of minicircles.Figure 7.


The replication of plastid minicircles involves rolling circle intermediates.

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

AFM images of minicircular DNA. Minicircular DNA of 6–8 kb fractions (A–G) and ∼3 kb fraction (H–J) were subjected to AFM. Images (A, E and G) were performed by scanning probe microscope from Veeco, while the rest (B–D, F and H–J) were by NanoScope III from Digital Instruments. The ethidium bromide stained gel on the left indicates the regions for DNA extractions (gDNA = genomic DNA of H. triquetra). The heights of objects in the AFM images can be denoted by the Z-scale on the right-handed top corner.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: AFM images of minicircular DNA. Minicircular DNA of 6–8 kb fractions (A–G) and ∼3 kb fraction (H–J) were subjected to AFM. Images (A, E and G) were performed by scanning probe microscope from Veeco, while the rest (B–D, F and H–J) were by NanoScope III from Digital Instruments. The ethidium bromide stained gel on the left indicates the regions for DNA extractions (gDNA = genomic DNA of H. triquetra). The heights of objects in the AFM images can be denoted by the Z-scale on the right-handed top corner.
Mentions: AFM is a useful tool to study topology of objects at the nanoscale. It has been used in biological research to image DNA and protein/DNA complexes (37,38). To directly investigate the shape of those double-sized minicircular DNA, AFM of the 6–8 kb fractions was performed. Minicircular DNA of the APBs as detected by Southern blot analysis was adsorbed on smooth surface mica and imaged with AFM. Figure 7 illustrates a number of images of thread-like structures, putative DNA species of minicircles.Figure 7.

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