On the 22 proteins, 18 proteins have been localized with the centromere throughout the mitotic cell cycle. Examination ples of this group are shown in Figure 1B. These proteins have been generally localized at just one spot all through interphase and as six spots in metaphase. Centro mere localization in the newly identi ed Cnl2 protein was examined by colocalization with Nuf2. More more, ChIP experiments con rmed that Cnl2 was localized in the central region,but not on the outer repeats,with the centromere. In contrast, 4 proteins had been localized at the centromere only in the M phase. These 4 proteins share homology together with the S. cerevisiae DASH com plex, DAM1, DAD2, ASK1, and SPC34,along with the observation that their centromere localization is restricted to your M phase is previously reported in S. pombe. As a result, we assigned these 4 proteins to your DASH complex.
Meiotic Behaviors of Kinetochore Proteins Observed in Residing Cells Subsequent we examined the meiotic behavior of those 22 centro mere proteins. In S. pombe, haploid met inhibitor cells within the opposite mating type conjugate on nitrogen starvation,two hap selelck kinase inhibitor loid nuclei fuse together through karyogamy, and meiotic prophase is characterized by the elongated horsetail nucleus stopping in the center within the cell, the nucleus condenses and initiates meiotic divisions. For the duration of nuclear movements, cen tromeres are separated from your SPB. Eighteen group one proteins behaved in a different way through meiosis and were even more classi ed into two subgroups, the Mis6 like and NMS groups. Proteins within the Mis6 like group remained at the centromere during meiosis,whereas those on the NMS group disappeared through the centromere or their presence was signi cantly diminished, dur ing meiotic prophase. The Mis6 like group incorporates nine proteins that behave like Mis6.
Amid these proteins, 6 proteins were found to get a part of a Mis6 containing complex isolated by biochemical puri cation. Cnp1 and Cnp3/ Mif2 are homologues of metazoan CENP A and CENP C, respectively. S. pombe Dad1 was localized in the centromere throughout mitosis as proven previously. Dad1 remained on the centromere all through meiosis at the same time as mitosis and was consequently classi ed like a Mis6 like group protein, regardless of the

fact that Dad1 belongs on the DASH complicated in S. cerevisiae. We classi ed Cnl2 and Fta7 as members of this group given that they remained on the centromere all through mitosis and meiosis. Moreover, their centromere localization depended on Mis6, Cnl2 and Fta7 proteins lost their centromere localization within a mis6 302 tem perature sensitive mutant at the limited temperature of 36 C. The NMS group is made up of nine proteins that disappear through the centromere through meiotic prophase. This group of proteins correspond to your biochemi cally de ned NMS supercomplex, which is comprised of your Ndc80 complicated,the Mis12 complicated,and Spc7.

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