Tsao D A, Wu C W, Lin Y S
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Gene. 1998 Oct 9;221(1):51-8. doi: 10.1016/s0378-1119(98)00432-6.
Telomerase is a ribonucleoprotein reverse transcriptase essential for the maintenance of telomere length. However, the available information concerning the biochemical and structural aspects of mammalian telomerases is scarce, primarily due to the low abundance of these enzymes and the difficulty and expense involved in its purification. To overcome these problems, we started to purify and characterize telomerase from bovine testis. Bovine telomerase was purified over columns of hydroxyapatite, DEAE-Sepharose, heparin-agarose, phenyl-agarose and spermine-agarose. In a sedimentation study performed using a 15-40% glycerol gradient, partially purified bovine telomerase exhibited an apparent molecular weight of more than 1000 kDa. One 435-bp RNA, bTR, was cloned from the most purified telomerase fraction and shown to be co-purified with telomerase activity in a glycerol gradient. bTR shares 83% similarity to the human telomerase RNA and 65% to the mouse telomerase RNA. A putative template region encompassing 10 nucleotides (5'-CUAACCCUAA-3') complementary to the mammalian telomere sequence (TTAGGG)n is located between nucleotides 38-47 of bTR. Besides, the bTR 5'-flanking region shares only three regulatory elements with that of hTR: a TATA-like sequence, a CCAAT box and an E1A-F box. Furthermore, the addition of in-vitro transcribed bTR reconstituted telomerase activity after removal of the endogenous bTR by micrococcal nuclease digestion. Northern blot analysis identified a single RNA of about 430-440 nucleotides expressed in the bovine testis and an immortalized bovine cell line. Taken together, these data strongly suggest that bTR is the RNA component of bovine telomerase.
端粒酶是一种核糖核蛋白逆转录酶，对维持端粒长度至关重要。然而，关于哺乳动物端粒酶生化和结构方面的现有信息很少，主要是因为这些酶的丰度低，以及纯化过程涉及的难度和费用。为了克服这些问题，我们开始从牛睾丸中纯化和表征端粒酶。牛端粒酶通过羟基磷灰石柱、DEAE-琼脂糖柱、肝素-琼脂糖柱、苯基-琼脂糖柱和精胺-琼脂糖柱进行纯化。在使用15%-40%甘油梯度进行的沉降研究中，部分纯化的牛端粒酶显示出大于1000 kDa的表观分子量。从最纯的端粒酶组分中克隆出一个435 bp的RNA，即bTR，并显示在甘油梯度中与端粒酶活性共纯化。bTR与人类端粒酶RNA有83%的相似性，与小鼠端粒酶RNA有65%的相似性。一个包含10个核苷酸（5'-CUAACCCUAA-3'）的推定模板区域，与哺乳动物端粒序列（TTAGGG）n互补，位于bTR的38-47核苷酸之间。此外，bTR的5'-侧翼区域与hTR的5'-侧翼区域仅共享三个调控元件：一个类TATA序列、一个CCAAT盒和一个E1A-F盒。此外，通过微球菌核酸酶消化去除内源性bTR后，添加体外转录的bTR可重建端粒酶活性。Northern印迹分析在牛睾丸和永生化牛细胞系中鉴定出一种约430-440个核苷酸的单一RNA。综上所述，这些数据强烈表明bTR是牛端粒酶的RNA组分。
