Supplemental Material

Movie S1-S6. Time-lapse movies of Mad2-GFP in cells shown in Figure 2 are provided. Images were taken every 30 seconds, and playback rate is 2 frames/second. For details, see the legend of Figure 2 and the Materials and Methods. S1, the wild type; S2, mis12-537 mutant; S3, cnp1-1 mutant; S4, mis6-302 mutant; S5, sim4+-shut-off; S6, nuf2-1 mutant.

This article contains the following supporting material:

• MovieS1.Mad2-GFP in the wild type - Mad2-GFP in the wild type
• MovieS2. Mad2-GFP in Mis12-537
• MovieS3. Mad2-GFP in cnp1-1
• Movie S4. Mad2-GFP in mis6-302
• Movie S5. Mad2-GFP in sim4-shut-off
• Movie S6. Mad2-GFP in nuf2-1
• Figure S1 Identification of fission yeast CENP-C homolog - (A) Sequence alignment of SpCENP-C with S. cerevisiae Mif2 (ScMif2) (Meluh and Koshland, 1995) and Homo sapiens CENP-C (HsCENP-C) (Saitoh et al., 1992). Identical amino acids conserved among 2 of 3 proteins are boxed. Systematic gene name of SpCENP-C designated by the S. pombe genome project is SPBC1861.01c.
  
  (B) Time-lapse images of SpCENP-C-GFP in a living wild type cell. The dynamic behavior of SpCENP-C-GFP during the cell cycle resembled that of the centromeres previously reported (Saitoh et al., 1997).
  
  (C) Wild type cells expressing either Sim4-GFP or SpCENP-C-GFP fusion were cultured in YES medium at 33?C and subjected to chromatin immunoprecipitation assay. The GFP tagged protein was immunoprecipitated, and co-precipitated DNA was amplified by the PCR method using the primers of cnt1, imr1, otr1 and lys1 (Takahashi et al., 2000). Approximately the same amount of PCR products was obtained from the whole cell extracts (WCE) of cells with or without GFP tagged proteins (Lanes 4, 5 and 6). Immunoprecipitates of GFP tagged proteins yielded the PCR products of cnt1 and imr1 (central core DNAs of centromere 1) but not of otr1 (pericentromeric heterochromatin DNAs of centromere 1) or lys1 (non-centromeric DNAs) (Lane 2 and 3), indicating that both Sim4 and SpCENP-C are specifically bound to the central centromeres (Takahashi et al., 1992). Lane 1 is the control Immunoprecipitate using the extract without GFP tagged proteins.