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Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat

View Article: PubMed Central

ABSTRACT

The kinetochore is a protein complex including kinetochore-specific proteins that plays a role in chromatid segregation during mitosis and meiosis. The complex associates with centromeric DNA sequences that are usually species-specific. In plant species, tandem repeats including satellite DNA sequences and retrotransposons have been reported as centromeric DNA sequences. In this study on sunflowers, a cDNA-encoding centromere-specific histone H3 (CENH3) was isolated from a cDNA pool from a seedling, and an antibody was raised against a peptide synthesized from the deduced cDNA. The antibody specifically recognized the sunflower CENH3 (HaCENH3) and showed centromeric signals by immunostaining and immunohistochemical staining analysis. The antibody was also applied in chromatin immunoprecipitation (ChIP)-Seq to isolate centromeric DNA sequences and two different types of repetitive DNA sequences were identified. One was a long interspersed nuclear element (LINE)-like sequence, which showed centromere-specific signals on almost all chromosomes in sunflowers. This is the first report of a centromeric LINE sequence, suggesting possible centromere targeting ability. Another type of identified repetitive DNA was a tandem repeat sequence with a 187-bp unit that was found only on a pair of chromosomes. The HaCENH3 content of the tandem repeats was estimated to be much higher than that of the LINE, which implies centromere evolution from LINE-based centromeres to more stable tandem-repeat-based centromeres. In addition, the epigenetic status of the sunflower centromeres was investigated by immunohistochemical staining and ChIP, and it was found that centromeres were heterochromatic.

No MeSH data available.


Immunostaining of sunflower metaphase chromosomes using an anti-HaCENH3 antibody. (A) DAPI-stained chromosomes. (B) Immunosignals of an anti-HaCENH3 antibody. (C) Merged image of (A,B). Scale bar, 10 μm.
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Figure 2: Immunostaining of sunflower metaphase chromosomes using an anti-HaCENH3 antibody. (A) DAPI-stained chromosomes. (B) Immunosignals of an anti-HaCENH3 antibody. (C) Merged image of (A,B). Scale bar, 10 μm.

Mentions: An anti-HaCENH3 antibody was raised against a synthetic peptide comprising N-terminal amino acid residues 2–21 of the deduced HaCENH3 amino acid sequence (Supplementary Image 1). To confirm its specificity to the centromeres, immunostaining using the antibody was conducted, and centromere-specific immunosignals appeared on all sunflower chromosomes (Figure 2). Additionally, in immunohistochemical staining, microtubule signals on all chromosomes were associated with all of the anti-HaCENH3 immunosignals at metaphase (Supplementary Movie 1).


Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat
Immunostaining of sunflower metaphase chromosomes using an anti-HaCENH3 antibody. (A) DAPI-stained chromosomes. (B) Immunosignals of an anti-HaCENH3 antibody. (C) Merged image of (A,B). Scale bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4628103&req=5

Figure 2: Immunostaining of sunflower metaphase chromosomes using an anti-HaCENH3 antibody. (A) DAPI-stained chromosomes. (B) Immunosignals of an anti-HaCENH3 antibody. (C) Merged image of (A,B). Scale bar, 10 μm.
Mentions: An anti-HaCENH3 antibody was raised against a synthetic peptide comprising N-terminal amino acid residues 2–21 of the deduced HaCENH3 amino acid sequence (Supplementary Image 1). To confirm its specificity to the centromeres, immunostaining using the antibody was conducted, and centromere-specific immunosignals appeared on all sunflower chromosomes (Figure 2). Additionally, in immunohistochemical staining, microtubule signals on all chromosomes were associated with all of the anti-HaCENH3 immunosignals at metaphase (Supplementary Movie 1).

View Article: PubMed Central

ABSTRACT

The kinetochore is a protein complex including kinetochore-specific proteins that plays a role in chromatid segregation during mitosis and meiosis. The complex associates with centromeric DNA sequences that are usually species-specific. In plant species, tandem repeats including satellite DNA sequences and retrotransposons have been reported as centromeric DNA sequences. In this study on sunflowers, a cDNA-encoding centromere-specific histone H3 (CENH3) was isolated from a cDNA pool from a seedling, and an antibody was raised against a peptide synthesized from the deduced cDNA. The antibody specifically recognized the sunflower CENH3 (HaCENH3) and showed centromeric signals by immunostaining and immunohistochemical staining analysis. The antibody was also applied in chromatin immunoprecipitation (ChIP)-Seq to isolate centromeric DNA sequences and two different types of repetitive DNA sequences were identified. One was a long interspersed nuclear element (LINE)-like sequence, which showed centromere-specific signals on almost all chromosomes in sunflowers. This is the first report of a centromeric LINE sequence, suggesting possible centromere targeting ability. Another type of identified repetitive DNA was a tandem repeat sequence with a 187-bp unit that was found only on a pair of chromosomes. The HaCENH3 content of the tandem repeats was estimated to be much higher than that of the LINE, which implies centromere evolution from LINE-based centromeres to more stable tandem-repeat-based centromeres. In addition, the epigenetic status of the sunflower centromeres was investigated by immunohistochemical staining and ChIP, and it was found that centromeres were heterochromatic.

No MeSH data available.