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First published online 28 September 2004
doi: 10.1242/jcs.01414

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Macroscopic folding and replication of the homogeneously staining region in late S phase leads to the appearance of replication bands in mitotic chromosomes

Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-hiroshima, 739-8521, Japan

View larger version (58K): [in a new window]   Fig. 1. Replication timing of plasmid repeats in the HSR. The procedure is diagrammed in (A). Logarithmically growing clone-22 cells were pulse-labeled with BrdU for 30 minutes and chased with fresh medium. At 4 (B), or 12 (C) hours after the BrdU labeling, metaphase spreads were prepared from the cultures. Incorporated BrdU (red) and the plasmid sequences (green) were simultaneously detected with an anti-BrdU antibody and FISH using a DIG-labeled plasmid probe, respectively. DNA was counterstained with DAPI in blue. When the cells were chased for 4 hours, the plasmid repeats in the HSR overlapped with the BrdU signal (yellow arrowhead in B). Both the entire metaphase image and the enlarged images are shown). When the cells were chased for 12 hours, the HSR did not overlap with the BrdU signal and appeared in light blue (white arrow in C). (D) The frequencies of metaphase spreads showing the BrdU signal at the chromosome arm () and at the HSR () are plotted against the chase time following BrdU pulse-labeling. (E,F) The time of replication of the HSR was compared with other late-replicating chromosomal domains. Eighty-six metaphases were classified into seven types according to the BrdU-labeling at the HSR (cyan), at the centromere (yellow arrows), and at the telomere (red arrows). The incidence of each type is summarized in E, where the colocalization of the BrdU signal with the HSR, centromere (Cen.), and telomere at the long arm (Telo.q) or short arm (Telo.p) is shown.

View larger version (70K): [in a new window]   Fig. 2. The HSR is composed of a homogeneous array of transfected plasmid sequences. (A-A'') Metaphase spreads prepared from clone-22 cells were simultaneously hybridized with the DIG-labeled plasmid probe and the biotinylated Alu probe; the hybridized probes were detected with different fluorescent colors. DNA was counterstained with DAPI. (A') The plasmid probe brightly and uniformly stained the HSR (two-headed arrow). (A'') At the HSR, the Alu probe never produced a signal, whereas it brightly labeled highly repetitive Alu sequences on the chromosomes. The signal appeared in bands at the chromosomes, because the Alu sequences were concentrated in the R-band (Matera and Ward, 1992). (B-B'') The metaphase was simultaneously hybridized with the DIG-labeled DM-painting micronuclei probe and the biotin-labeled plasmid probe. (B'') The first probe hybridized with the amplicon already present in the parental COLO 320DM cells, and it painted the DMs in clone-22 cells (compare arrowheads in B and B''), but it never hybridized to the HSR (compare two-headed arrow in B and B''). The DM-derived sequence flanking the HSR is indicated by an arrow (B'').

View larger version (106K): [in a new window]   Fig. 3. The HSR shows reproducible replication bands that depend on the HSR length. Clone-22 (A,B,E,F) and clone-24 cells (C,D,G,H) were analyzed in the same experiment as shown in Fig. 1H. The replication sites pulse-labeled in late S phase appeared as bands in the mitotic HSR. The arrows in A to D indicate the positions of the bands. The number of bands appeared to be proportional to the length of the HSR. A portion of the clone-22 and clone-24 cells had pairs of homologous HSRs. Such pairs within a single cell had similar replication bands (E-H). In E, the orientation of the HSR is shown as two arrows. For panels G and H, two microscope fields of the identical metaphase cell were digitally assembled to form one panel.

View larger version (73K): [in a new window]   Fig. 4. Progression of the replication bands analyzed by the double pulse-chase experiment. (A) Outline of the procedure. Clone-22 cells were pulse-labeled for 10 minutes with IdU, chased in fresh medium for varying times, pulse-labeled for 10 minutes with CldU, and further chased with fresh medium. The cells were harvested 4 hours after the IdU pulse-labeling, and subjected to metaphase spread preparation. The sites of IdU and CldU incorporation were detected using specific antibodies, and are shown in red and green pseudo-color, respectively. The HSR was detected by FISH using a biotinylated plasmid probe and Alexa 647-conjugated streptoavidin, which is shown in blue pseudo-color in this figure. The chase times between the IdU and CldU pulse-labeling are noted in the CldU panels. (B) Summary of the frequencies of HSRs labeled with both IdU and CldU at the varying interval time; (C-I) representative images. The orientation of the chromosomes detected with DAPI-staining (not shown) are indicated with thin lines.

View larger version (126K): [in a new window]   Fig. 5. Replication sites of the HSR subchromosomal domain in the late S phase nucleus. Clone-22 cells were pulse-labeled with BrdU for 30 minutes, and then the cells were immediately fixed with PFA. The site of BrdU incorporation was detected with an anti-BrdU antibody in red (A'-C'); HSR was detected by hybridization with a DIG-labeled plasmid probe in green; merged images in A-C. In whole nuclear images (left image of each panel), approximate nuclear rim was outlined based on the faint background of BrdU-signal (light blue circles). The images show that these nuclei were in late S phase, because the BrdU labeling was restricted to the internal large heterochromatin islands. Serial confocal images of 0.68-µm intervals in the z-axis were obtained for each HSR domain (right panels). There were three types of BrdU-labeling: (A) at the peripheral region, (B) at the whole domain and (C) at the internal region. Bars, 5 µm.

View larger version (85K): [in a new window]   Fig. 6. Progression of the replication sites of the HSR subchromosomal domain. Clone-22 cells were pulse-labeled with IdU for 10 minutes, chased for 40 minutes in fresh medium, and then pulse-labeled with CldU for 10 minutes. The cells were immediately fixed with PFA, and the site of IdU or CldU labeling was detected with specific antibodies, which are shown in red and green pseudo-color, respectively. The HSR was detected by FISH and is shown in blue pseudo-color. (A-C) Whole nuclear images show that cells were in late S phase. In the images, the approximate nuclear rim was outlined based on the faint background FISH-signal (cyan circles). The enlarged images for the HSR domain show that the replication site changed from the periphery of the HSR domain to the internal region during the IdU and CldU labeling. For clarity, two-color (IdU plus CldU) and three-color (IdU, CldU plus HSR) merged images are shown (A'-C' and A''-C'', respectively). Serial confocal images of 0.64-µm intervals in the z-axis that intercept the center of the HSR domain are shown. Arrowheads point to the replication of the telomere of the same arm as the HSR. Bars, 5 µm.

View larger version (57K): [in a new window]   Fig. 7. Shape of the HSR domain in the late G2 phase nucleus. The clone-22 cells were pulse-labeled with IdU, chased for 100 minutes, pulse-labeled with CldU, further chased, and harvested 4 hours after the IdU-labeling. The incorporated IdU or CldU was detected simultaneously with the HSR, as in Fig. 4. Representative images of the interphase nuclei are shown whose nuclei should be at the late G2 phase at the time of harvest, because the late-replicating HSR was labeled with CldU (A) or IdU (B). The HSR detected by FISH is (A) ring-shaped or in an (B) extended pair of spirals. Arrows indicate HSR (A',B') labeled with CldU (A''') or IdU (B'').

View larger version (26K): [in a new window]   Fig. 8. A coiled-coil model for the folding and replication of the HSR. A long and homogeneous array of repeat sequences might be folded as a giant coil that is further coiled `coiled coil' (A-C). When drawing the unit structure of replication firing, we tentatively assumed that one 30-nm fiber (shown in thin lines) might be folded into a replicon cluster. The initiation of replication (red line) from the periphery of the coiled coil would appear as replication bands in the mitotic chromosome (A'-C'). Topologically, the peripheral region might be loose and the inner region might be packed tightly, offering an explanation why replication starts in the periphery. To replicate the inner region, the entire coil might be loosened at the last stage (C).

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