Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome
Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome
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DOI：
10.1083/jcb.201106036
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发表时间：
2011-11-14
影响因子：
7.8
通讯作者：
Salmon, E. D.
中科院分区：
文献类型：
--
作者：
Lawrimore, Josh;Bloom, Kerry S.;Salmon, E. D.
Cse 4是CENP-A的芽殖酵母同源物，CENP-A是一种修饰的组蛋白H3，其指定所有真核生物中动粒的碱基。芽殖酵母的独特之处在于每个着丝粒只有一个着丝粒微管附着位点。着丝粒由CEN DNA、序列特异性结合复合物（CBF 3）和含Cse 4的核小体指定。在这里，我们比较的比率动粒近端Cse 4-GFP荧光在后期的几个标准，包括纯化的EGFP分子在体外产生的GFP融合蛋白的拷贝数的校准曲线。我们的结果得出一个平均值。类似于5个Cse 4s，类似于3个内动粒CBF 3复合物，和。类似于20个外动粒Ndc 80复合物。我们的校准测量增加了2.5-3倍的蛋白质拷贝数在真核动粒的基础上，假设两个Cse 4每个芽殖酵母动粒先前的比率测量。所有大约五个Cse 4s可能与CEN核小体，但我们表明，平均三个Cse 4s可以位于侧翼核小体在染色体之间不同的随机位点。
Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of. similar to 5 Cse4s, similar to 3 inner kinetochore CBF3 complexes, and. similar to 20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.