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. 2013 Jan 7;200(1):45–60. doi: 10.1083/jcb.201210106

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© 2013 Hori et al.

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Figure 3. — Characterization of the CENP-C–derived kinetochores. (A) Diagram showing the chicken CENP-C sequence and the tested deletion constructs for the LacO–LacI–based kinetochore induction assay shown in Fig. 1. The yellow box shows a putative binding domain with the Mis12 complex. Mif2 II and Mif2 III show homology regions with yeast Mif2 (red). The survival rate of cells after removal of native centromere of chromosome Z in cells expressing each LacI fusion construct with multiple deletions is shown in the diagram (right). The survival rate was increased in cells expressing LacI fusion with CENP-C (1–643) or CENP-C (601–864). The assay was completed twice for CC full-length, once for CC^1–643, once for CC^74–643, once for CC^1–165, twice for CC^601–761, and three times for CC^601–864. (B) Growth curve of CC^601–864-LacI/ΔZcen cells in the presence (red line) or absence of IPTG (black line). This measurement was completed once in each condition. (C) Localization of CENP-A, CENP-C, CENP-T, and Ndc80 at the LacO locus in CC^601–864-LacI/ΔZcen cells. These experiments were performed in the absence of IPTG. Arrows indicate red signals stained with each antibody. Zq, Z chromosome. (D) CENP-A–associated DNAs isolated by ChIP with anti–CENP-A antibodies from CC^601–864-LacI/ΔZcen cells were hybridized with a probe containing the LacO sequence or a centromere DNA from chromosome 5. (E) Growth curve of CC^1–643-LacI/ΔZcen cells in the presence or absence of IPTG. This measurement was completed once in each condition. (F) Representative images of lagging chromosome Z stained by the Z-specific probe (red) during anaphase of CC^1–643-LacI/ΔZcen cells after addition of IPTG. (G) Examination of loss or gain of chromosome Z after addition of IPTG to CC^1–643-LacI/ΔZcen cells (top). As a control, numbers of chromosome 1 were also scored (bottom). Chromosome Z was lost in >80% of cells at 48 h after the addition of IPTG. Loss or gain of chromosome 1 was not observed after addition of IPTG to CC^1–643-LacI/ΔZcen cells. This experiment was completed once in each condition (CC^1–643-LacI/+Zcen +IPTG, n = 229; CC^1–643-LacI/ΔZcen 0 h, n = 281; 24 h, n = 235; 48 h, n = 204). (H) CENP-A–associated DNAs isolated by ChIP with anti-CENP-A antibodies from CC^1–643-LacI/ΔZcen cells were hybridized with a probe containing the LacO sequence or a centromere DNA from chromosome 5. LacO sequences were not detected in CENP-A immunoprecipitates in CC^1–643-LacI/ΔZcen cells. (I) Localization of Mis12 at the LacO locus in cells expressing LacI fusions with CENP-C (1–643), CENP-C (74–643), or CENP-C (1–165). Mis12 signals were detected in cells expressing LacI fusion with CENP-C (1–643), but not detected in cells expressing LacI fusions with CENP-C (74–643) or CENP-C (1–165). Arrows indicate the position of the LacO sequence. Z, Z chromosome.