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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α DNA的特异性识别。着丝粒区的克隆。Alphoid DNA家族是位于人类染色体着丝粒区的一种高度重复的DNA序列。170 bp长的α重复序列的阵列似乎延伸超过几百kb。用限制性内切酶BglII消化染色体DNA产生约200 kb长的α DNA片段，通过在蔗糖密度梯度中离心，可以将其与来自染色体其它区域的较小DNA片段分离。为了克隆长α DNA，并在人培养细胞中检测其生物学活性，我们用S.通过插入潮霉素抗性基因和ARS（自主复制序列）构建酿酒酵母。利用脉冲场凝胶电泳和Southern杂交对pYAC文库的酵母转化子进行了长α重复序列的检测.着丝粒区的DNA-蛋白质相互作用。我们已经显示了一种人类着丝粒抗原蛋白，其通过自身免疫性疾病患者的抗血清检测到与α DNA特异性相互作用。着丝粒抗体免疫沉淀或凝胶迁移试验表明，80 kD的CENP-B蛋白结合的17 bp长的DNA基序存在于α重复。DNA序列分析表明，除Y染色体外，所有染色体上都存在17 bp的基序（CENP-B box）。因此，CENP-B蛋白与CENP-B盒之间的相互作用可能参与了着丝粒在染色体分离中的作用。