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Analysis and clinical application of telomerase and telomere functions

端粒酶和端粒功能分析及临床应用

基本信息

批准号：
12470501
负责人：
IDE Toshinori
金额：
$ 8.9万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470501/
关键词：
telomere telomere DNA extra chromosomal telomere repeats ECTR telomere-binding proteins telomerase immortalization of cell proliferative life span

项目摘要

Telomere is specialized structure at the end of eukaryotic chromosomes which functions in chromosome stability, positioning, replication and meiosis. Telomere is not composed of common nucleosomal structure but of a complex with several types of telomere specific proteins. Absence of adequate techniques to separate telomeres from non-telormeric parts of chromosome fails to clarify precise structure of telomeres. We found that telomerase-negative immortal human cells contained extra-chromosomal telomere repeats (ECTR) and ECTR was complexed with one of telomere-binding protein, TRF-1. We tried here separation of ECTR protein complex from the bulk chromosome and analysis of protein components in this complex. KMST-6, a human cells line that contains a large number of ECTR, was collected at mitotic phase of the cell cycle and lysed in the buffer containing mild detergent. ECTR-protein complex was separated by differential centrifugation and density gradient centrifugation. When 1 % of total DNA was recovered in ECTR-protein complex fraction, this fraction contains 10 % of total telomere sequence and 1.5 % of total Alu sequence. Further purification by separating nucleosomal chromatin using anti-histone antibody was failed because antibodies were not good for immunoadsorption.

端粒是真核染色体末端的特殊结构，在染色体稳定性、定位、复制和减数分裂中发挥作用。端粒不是由常见的核小体结构组成，而是由几种端粒特异性蛋白质的复合物组成。由于缺乏足够的技术将端粒与染色体的非端粒部分分开，因此无法阐明端粒的精确结构。我们发现端粒酶阴性的永生人类细胞含有染色体外端粒重复序列（ECTR），并且ECTR与端粒结合蛋白之一TRF-1复合。我们在这里尝试从大量染色体中分离 ECTR 蛋白质复合物，并分析该复合物中的蛋白质成分。 KMST-6 是一种含有大量 ECTR 的人类细胞系，在细胞周期的有丝分裂期收集并在含有温和去污剂的缓冲液中裂解。 ECTR-蛋白质复合物通过差速离心和密度梯度离心分离。当 ECTR-蛋白质复合物级分中回收到总 DNA 的 1% 时，该级分包含总端粒序列的 10% 和总 Alu 序列的 1.5%。使用抗组蛋白抗体分离核小体染色质进一步纯化失败，因为抗体不利于免疫吸附。