Cloning of Large Segments from the Centromere Region of Human Chromosome

为了研究人类染色体着丝粒的结构和功能，我们一直试图从着丝粒区域分离出大的DNA片段。另一方面，另一种研究DNA-蛋白质相互作用的方法揭示了着丝粒抗原蛋白对着丝粒αDNA的特异性识别。着丝粒区域的克隆。阿尔菲德DNA家族位于人类染色体着丝粒区域，是一个高度重复的DNA序列。长达170个碱基对的阿尔菲重复序列似乎延伸了超过几百kb。用限制性内切酶BglII对染色体DNA进行酶切，可得到约200kb的Alphid DNA片段，并可通过蔗糖密度梯度离心法将其与来自染色体其他区域的较小DNA片段分离。为了克隆长链霉菌DNA并检测其在人培养细胞中的生物学活性，通过插入潮霉素抗性基因和自主复制序列，构建了酿酒酵母pYAC质粒的衍生体。利用脉冲场凝胶电泳法和Southern杂交技术对pYAC文库的酵母转化子进行了长Alphid重复序列的检测。着丝粒区域的DNA-蛋白质相互作用。我们已经展示了一种由自身免疫性疾病患者的抗血清检测到的人类着丝粒抗原蛋白，它与Alphid DNA特异地相互作用。着丝粒抗体免疫沉淀法或凝胶移位实验表明，80kD的CENP-B蛋白与存在于ALPHIY重复序列中的17bp长的DNA基序结合。DNA序列分析表明，除Y染色体外，所有染色体均含有17bP基序(CENP-B盒)。因此，CENP-B蛋白与CENP-B盒的相互作用可能参与着丝粒在染色体分离中的作用。

To examine the structure and function of the centromere of human chromosome, we have been trying to isolate large DNA fragments from the centromere region. On the other hand, another approach to investigate DNA-protein interaction revealed the specific recognition of a centromeric alphoid DNA by a centromere antigenie protein.1. Cloning of the centromere region. Alphoid DNA family, a highly repeated DNA sequence, is located at the centromere region of human chromosome. The array of 170 bp long alphoid repeats seems to extend more than several hundreds kb. Digestion of chromosomal DNA with the restriction enzyme BglII yielded about 200 kb long alphoid DNA fragments, which can be separated from smaller DNA fragments derived from other regions of chromosome by centrifugation in a sucrose density gradient. To clone long alphoid DNA and then to examine its biological activity in human cultured cell, a derivative of pYAC plasmid of S. cerevisiae was constructed by inserting the hygromycine-resistant gene and an ARS (autonomously replicating sequence). Yeast transformants of pYAC library are under investigation for the presence of long alphoid repeats by using pulse field gel electrophoresis and southern hybridization.2. DNA-Protein interaction in the centromere region. We have shown one of the human centromere antigenic proteins that are detected by antisera of autoimmune disease patients specifically interacts with alphoid DNA. Immunoprecipitation using centromere antibody or gel-shift assay revealed that the 80 kD CENP-B protein binds to 17 bp long DNA motif present in alphoid repeats. Analysis of DNA sequence of the cloned alphoid sequence shows that the 17 bp motif (CENP-B box) is present in all the chromosome except for Y- chromosome. Therefore, it is likely that the interaction between CENP-B protein and the CENP-B box participates in the function of the centromere in the segregation of chromosome.