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. 2008 Mar 24;180(6):1115–1131. doi: 10.1083/jcb.200708170

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Copyright © 2008, The Rockefeller University Press

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Figure 2. — Cnp1, Mis6, and Mis13 are required for localizing condensin at the kinetochore. (A) Cells cultured at 26°C in EMM2 were observed for the colocalization of Cnd1-GFP with Mis12-RFP, a centromere/kinetochore protein. The numbers in the right panels indicate time in minutes. The enlarged images of Cnd1-GFP (left), Mis12-RFP (middle), and the merged images (right) at 13 min are shown in the insets. (B) Cut14-GFP and Sad1-RFP were observed in the wild-type, mis6-302, cnp1-1, mis13-1, mis16-53, and mis18-262 mutants cultured at 26°C and were shifted to 36°C for 8 h. (C) Chromosomally integrated Cnp1/CENP-A–GFP expressed under the native promoter was observed in the wild-type and cut14-208 mutant cultured at 36°C for 2 h. (D) A ChIP assay was performed using extracts of block-released nda3-311 mutant that expressed Cut14-Flag. The probes were from the central centromere, cnt1 (c10, c9, and c7.5), imr1 (c4 and c1), the outer centromere dg, and the noncentromeric lys1 ⁺. WCE, whole cell extract; IP, immunoprecipitate. Quantitative data are shown at the bottom (see Results for details). (E) A ChIP assay was performed using extracts of block-released cdc25-22 mutant that expressed Cut14-Flag. Extracts of cdc25 cells (blocked in G2 at 36°C and released to 26°C) were prepared at 0 min (G2 phase), 25 min (prometaphase), 50 min (meta/anaphase), and 75 min (telophase or G1/S phase). The probes used were from the central centromere, cnt1 (c9), imr1 (c4), and the three arm probes arm1 (SPBC28F2.08c coding region), arm2 (SPBC29B5 noncoding region), and arm3 (ars2004). In the bottom panel, the percent frequencies of different types of cells are indicated.(F) A ChIP assay was performed using extracts of asynchronous wild-type, mis6-302, and cnp1-1 mutants that expressed Cut14-Flag. These strains were cultured at 26°C and were shifted to 36°C for 8 h. Real-time PCR was performed using the primers of cnt1, imr1, lys1 ⁺, and rDNA (N1). The levels of precipitated cnt1 and imr1 were diminished in cnp1-1 and mis6-302 mutants. Because the asynchronous cultures were used, the differences between wild-type and mutant cells were relatively small. Error bars represent SD. Bars: (A and C) 10 μm; (B) 2 μm.