Cloning of Large Segments from the Centromere Region of Human Chromosome

人类染色体着丝粒区域大片段的克隆

基本信息

批准号：
63580204
负责人：
MASUKATA Hisao
金额：
$ 1.6万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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.

为了检查人类染色体的丝粒的结构和功能，我们一直在试图将大型DNA片段与丝粒区分离。另一方面，另一种研究DNA蛋白相互作用的方法揭示了通过丝粒抗原蛋白对丝粒字母DNA的特异性识别。1。中心粒区的克隆。高度重复的DNA序列的字母DNA家族位于人类染色体的中心区域。 170 bp的长字母重复序列的阵列似乎延伸了数百个KB。用限制酶BGLII对染色体DNA的消化产生了约200 kb的长字母DNA片段，可以将其与蔗糖密度梯度以离心的其他染色体区域得出的较小DNA片段分离。为了克隆长的字母DNA，然后检查其在人培养细胞中的生物学活性，通过插入抗湿霉素耐药基因和ARS（自主复制序列）来构建酿酒酵母Pyac质粒的衍生物。 PYAC文库的酵母转化体正在研究使用脉冲场凝胶电泳和南部杂交的长α重复序列的研究。2。中心粒区域的DNA蛋白相互作用。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丝粒抗原蛋白之一，该抗原蛋白与alphoid DNA特别相互作用。使用CentRomere抗体或凝胶迁移测定法的免疫沉淀表明80 kD CENP-B蛋白与α重复序列中存在的17 bp长的DNA基序结合。克隆的字母序列的DNA序列的分析表明，除Y-染色体外，所有染色体都存在17个BP基序（CENP-B框）。因此，CENP-B蛋白与CENP-B盒之间的相互作用很可能参与了丝粒染色体隔离中的丝粒的功能。