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First published online 25 May 2004
doi: 10.1242/jcs.01144

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Differential localization of the centromere-specific proteins in the major centromeric satellite of Arabidopsis thaliana

¹ Core Research for Evolutionary Science and Technology, Japan Science and Technology, Kawaguchi 332-0012, Japan
² Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan

View larger version (57K): [in a new window]   Fig. 1. FISH images of A. thaliana cell cultures probed with 180 bp repetitive sequence (A-H) and with Athila (G,H). Chromosomes and nuclei were counterstained with DAPI (blue). The 180 bp repetitive sequences are shown in red (A-F) or green (G,H) and Athila as red (G,H). A-F show the following features: (A) the smallest chromosome found (arrow) and the stretched end of SAT-chromosome (arrowhead); (B) a ring-shaped chromosome (arrow); (C) a putative dicentric chromosome with two distinct 180 bp signals (arrow); (D) two chromosomes with two distinct and minor multiple 180 bp signals (arrows); (E) chromosomes carrying very small amounts of 180 bp repeats (arrows); (F) the same metaphase cell as in E showing only the 180 bp signals (enhanced to be visualized clearly). (G,H) A prometaphase (G) and an interphase (H) cell, probed with 180 bp repeats (green) and with Athila (red). Scale bars: 5 µm.

View larger version (64K): [in a new window]   Fig. 2. Immunofluorescence labeling of HTR12 and FISH detection of 180 bp repeats and Athila. (A-C) Immunosignals of HTR12 (red), FISH signals of 180 bp repeats (blue) and Athila (green) on prometaphase chromosomes (A), metaphase chromosomes (B) and in an interphase nucleus (C). Scale bars: 5 µm. (D-F) HTR12, 180 bp and Athila signals on the three (as in A-C) chromosomes, and the superimposed images. Scale bars: 1 µm. (G,H) HTR12 (red) and 180 bp repetitive sequence (blue) on two different sized prometaphase chromosomes (G) and in an interphase nucleus (H). Arrows indicate completely overlapping signals. Scale bars: 1 µm.

View larger version (67K): [in a new window]   Fig. 3. Immunofluorescence labeling of phosphorylated histone H3 and HTR12 on putative dicentric chromosomes. Chromosomes were counterstained with DAPI (blue). (A) Immunosignals from phosphorylated histone H3 (red), (B) FISH signals from 180 bp repetitive sequence (green) on a dicentric chromosome, (C) superimposition of A and B. Scale bar: 2 µm. (D) A metaphase cell containing a putative dicentric chromosome (arrow). HTR12 and two 180 bp repeat sites appear as red and green, respectively. Scale bar: 2 µm. (E) An anaphase cell with chromatin bridges. Arrows indicate HTR12 signals (red) and FISH signals of 180 bp repeats (green). Scale bar: 5 µm.

View larger version (87K): [in a new window]   Fig. 4. Immunofluorescence labeling of HTR12, and FISH detection of 180 bp repetitive and Athila sequences on extended chromatin fibers. (A) Immunosignals from HTR12 (red) on DAPI-staining extended chromatin fibers (white). (B) FISH signals from 180 bp repeats (green). (C) Superimposition of the HTR12 signals in A and the image in B, with overlapping regions shown in yellow. (D) FISH signals from Athila (blue). (E) Superimposition of HTR12 signals in A and the image in D. (F) Superimposition of 180 bp repeat signals in B and the image in E. Scale bar: 5 µm. (G) Immunosignals from HTR12 and FISH signals from 180 bp repeats on fully extended chromatin fibers. The knobs of 180 bp repeats are colocalized (yellow) with immunosignals of HTR12. Scale bar: 5 µm.

View larger version (101K): [in a new window]   Fig. 5. Immunolabeling of anti-AtCENP-C and anti-HTR12, and FISH with 180 bp repeats as a probe. Immunosignals of anti-AtCENP-C (green) and anti-HTR12 (red) and FISH signals of 180 bp repeat (blue) detected on a prometaphase cell (A), a metaphase cell (B) and an interphase cell (C). Counterstaining of chromosomes or nucleus with DAPI appears gray. Scale bars: 5 µm.

View larger version (93K): [in a new window]   Fig. 6. Immunofluorescence labeling of phosphorylated histone H3 and HTR12, and FISH detection of 180 bp repetitive sequences on mitotic metaphase chromosomes. (A-C) Polar view of chromosomes spread on the equatorial plate. (D-G) Equatorial view of chromosomes arranged on the equatorial plate. (A,E) FISH signals from 180 bp repeats (blue), (B,F) FISH signals from 180 bp repeats (blue) and immunosignals from phosphorylated histone H3 (green), (C,G) immunosignals from phosphorylated histone H3 (green) and from HTR12 (red). Chromosomes counterstained with DAPI appear gray (A-C) and pale gray (D). Scale bars: 2 µm.