Masumoto H, Masukata H, Muro Y, Nozaki N, Okazaki T
Department of Molecular Biology, School of Science, Nagoya University, Japan.
J Cell Biol. 1989 Nov;109(5):1963-73. doi: 10.1083/jcb.109.5.1963.
We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.
我们报道了一种人类着丝粒抗原与α卫星DNA（一种人类着丝粒卫星DNA，由170个碱基对的重复单元组成）之间的相互作用。用硬皮病患者的抗着丝粒血清可选择性免疫沉淀与HeLa细胞核提取物一起孵育的克隆α卫星DNA片段。通过引物延伸对DNA进行免疫沉淀，确定了α卫星DNA上形成DNA-抗原复合物所需的17个碱基对片段。另一方面，当通过链霉亲和素琼脂糖回收与携带17个碱基对基序的生物素化α卫星DNA结合的蛋白质并进行免疫印迹时，可检测到80kD的着丝粒抗原（CENP-B）。对用抗着丝粒血清免疫沉淀、经凝胶电泳分离并转移到膜上的蛋白质进行DNA结合实验，有力地表明80kD抗原特异性结合具有17个碱基对基序的DNA片段。这个17个碱基对的基序被称为“CENP-B盒”。除了迄今为止克隆的源自Y染色体的α卫星单体外，在所有已知的α卫星亚类中都发现了带有CENP-B盒的α卫星单体，其频率各不相同。这些结果表明,80kD着丝粒抗原与α卫星重复序列中的CENP-B盒之间的相互作用可能在人类着丝粒特定结构和/或功能的形成中发挥关键作用。
