Analysis and clinical application of telomerase and telomere functions

端粒是真核生物染色体末端的特殊结构，在染色体的稳定、定位、复制和减数分裂中起作用。端粒不是由共同的核小体结构组成，而是由几种类型的端粒特异性蛋白质组成的复合物。缺乏足够的技术来分离染色体的端粒和非端粒部分未能阐明端粒的精确结构。我们发现端粒酶阴性的不朽人细胞含有染色体外端粒重复序列（ECTR）， ECTR与端粒结合蛋白之一TRF-1络合。我们在此尝试了从体染色体中分离ECTR蛋白复合体并分析该复合体中的蛋白质成分。KMST-6是一种含有大量ECTR的人细胞系，在细胞周期的有丝分裂期收集，并在含有温和洗涤剂的缓冲液中裂解。采用差速离心和密度梯度离心分离ectr -蛋白复合物。当ECTR-protein复合物片段中总DNA的1%被回收时，该片段包含总端粒序列的10%和总Alu序列的1.5%。使用抗组蛋白抗体分离核染色质的进一步纯化失败，因为抗体不适合免疫吸附。

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.