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Grover, G. J. and Garlid, K. D.
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Han, J., Kim, N., Kim, E., Ho, W. and Earm, Y. E.
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Hanley, P. J., Mickel, M., Loffler, M., Brandt, U. and Daut, J.
Fig. 1.

Mitotic fission yeast centromeres are organized in cytologically separable domains. (A) Schematic diagrams of centromere structures. Centromere 1 is shown as an example but centromeres 2 and 3 have similar symmetric organization. Top: centromere 1 DNA consists two regions containing a region of centromeric outer repeats ( and ) flanking a single central core region containing the sequence. Bottom: a putative structure of centromere 1 in sister centromeres at metaphase (based on data in Fig. 1B,C,E and supplemental data at http://jcs.biologists.org/supplemental/ ). Kinetochores (Kin) are separated and facing opposite spindle poles whereas the regions are held together by sister-chromatid cohesion. The central core regions are the bases for Kin structures. (B) Deconvolved IF images of fixed metaphase cells showing two pairs of sister kinetochores. The DNA sequences were detected by FISH (green) and are flanked by the kinetochore protein (Ndc80) signal (red). (C) Frames from live imaging movies (see Movies 1 and 2 at http://jcs.biologists.org/supplemental ) showing the spindle pole body protein Cut12-CFP (red) along with Ndc80-GFP (green) in one cell (left) and Cut12-GFP (red) along with CFP-Cnp1 (green) in another cell (right). Bottom panel: the graphs show the pixel intensities along the spindle axis in the cells of Cut12 (red) and Cnp1 or Ndc80 as indicated (green). The length of the spindle axis is indicated by large black arrowed lines below each graph and the distance between two adjacent Ndc80 (0.41 μm) or Cnp1 (0.25 μm) signals on the spindle axis are indicated by small black arrowed lines. (D) Top: frames from the movie showing Ndc80-GFP (green) along with CFP-Cnp1 and Cut12-CFP (red) in one cell. The dotted squares represent the pixel position of the Ndc80 signals (see Movies 1 and 2 at http://jcs.biologists.org/supplemental ). Bottom: pixel shift control of the Hu1048 strain expressing the same protein in dual colors: Cut12-CFP (green) and Cut12-GFP (red). The perfect colocalization of the two colors of Cut12 (bottom) shows that the pixel differences between Cnp1 and Ndc80 (top) are significant. (A-C) Scale bar: 0.50 μm.

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Media were prepared according to standard methods ( Merrell ALL OUT BLAZE SIEVE Walking sandals vineyard wine nOCE2
). Strains used are listed in Table 1 . Strains carrying genes fused to green and cyan fluorescent proteins (GFP and CFP) were constructed using the methods described previously ( Bahler et al., 1998 ) with the following modifications. Plasmids containing CFP were constructed by replacing the entire coding region of GFP with that of CFP using standard techniques. Briefly, CFP was amplified by PCR using the cameleon pYC2 as template ( Miyawaki et al., 1997 ) and subsequently ligated into pFA6 in which GFP had been released. Expression of N-terminally tagged genes was controlled by the thiamine repressed nmt1 promoter.

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