The centromere-kinetochore complex: a repeat subunit model.
The centromere-kinetochore complex: a repeat subunit model.
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DOI：
10.1083/jcb.113.5.1091
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发表时间：
1991-06
期刊：
影响因子：
--
通讯作者：
Brinkley BR
中科院分区：
文献类型：
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作者：
Zinkowski RP;Meyne J;Brinkley BR
使用两种新技术研究了着丝粒的三维结构和着丝粒-着丝粒区域的DNA/蛋白质组成：咖啡因诱导未复制的着丝粒的分离以及通过细胞离心机中产生的低渗和/或剪切力拉伸着丝粒。通过 EM 和 CREST 自身抗体免疫染色证实着丝粒脱离。对连续切片的电子显微镜分析表明，分离的动粒代表来自整个动粒的片段。这对于雄性印度麂的七个大动粒来说尤其明显，在脱离时会产生 80-100 个碎片。所有着丝粒片段都与纺锤体微管相互作用，并在整个有丝分裂运动中进行，为着丝粒内的亚基组织提供了证据。通过低渗处理拉伸或解开中期着丝粒-着丝粒复合体，获得了对重复亚基模型的进一步支持。当用 CREST 自身抗体进行免疫染色并随后使用合成着丝粒探针进行原位杂交处理时，拉伸的动粒显示出沿着丝粒 DNA 纤维以重复模式排列的荧光亚基的线性阵列。除了 CREST 抗原之外，还发现每个重复亚基都与微管蛋白结合，并含有细胞质动力蛋白（一种位于冠状区域的微管马达）。总的来说，数据表明，动粒（EM 在许多真核染色体上看到的板状结构）是由线性 DNA 纤维折叠形成的，该纤维由串联重复的亚基组成，散布着 DNA 连接体。与之前提出的任何模型不同，该模型可以解释着丝粒的结构和进化多样性及其与许多物种真核染色体着丝粒的关系。
The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.