Lin J J, Zakian V A
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
Cell. 1995 Jun 30;81(7):1127-35. doi: 10.1016/s0092-8674(05)80017-0.
Telomerase activity was demonstrated in cell-free extracts from S. cerevisiae through the use of a PCR-based assay. As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP. Telomerase was not detected in extracts prepared from cells grown for approximately 30 or more cell divisions in the absence of the EST1 product, Est1p. TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1 delta cells. Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p. These data indicate that Est1p is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.
通过基于PCR的分析方法，在酿酒酵母的无细胞提取物中证实了端粒酶活性。正如预期的那样，这种活性通过对提取物进行RNase或苯酚处理而被消除，并且依赖于dGTP和dTTP。在缺乏EST1产物Est1p的情况下生长约30次或更多次细胞分裂的细胞制备的提取物中未检测到端粒酶。TLC1 RNA在体内决定端粒DNA的序列，在est1δ细胞中含量正常。此外，TLC1 RNA与表位标记的Est1p特异性沉淀。这些数据表明，Est1p要么是酵母端粒酶的一个亚基，要么是与端粒酶相关的辅助蛋白，在体外对其活性至关重要。
