Analysis and clinical application of telomerase and telomere functions

端粒是真核生物染色体末端的特化结构，在染色体稳定、定位、复制和减数分裂中起重要作用。端粒不是由普通的核小体结构组成，而是由几种端粒特异性蛋白组成的复合体。由于缺乏足够的技术来分离端粒和染色体的非端粒部分，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）和ECTR与端粒结合蛋白之一，TRF-1复合。我们尝试从大量染色体中分离ECTR蛋白复合物并分析该复合物中的蛋白组分。在细胞周期的有丝分裂期收集含有大量ECTR的人细胞系KMST-6，并在含有温和去污剂的缓冲液中裂解。采用差速离心和密度梯度离心分离ECTR-蛋白复合物。当在ECTR-蛋白质复合物级分中回收1%的总DNA时，该级分含有10%的总端粒序列和1.5%的总Alu序列。用抗组蛋白抗体分离核小体染色质的进一步纯化失败，因为抗体不利于免疫吸附。

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.