Prowse K R, Avilion A A, Greider C W
Cold Spring Harbor Laboratory, NY 11724.
Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1493-7. doi: 10.1073/pnas.90.4.1493.
Telomerase activity was identified in extracts from several different mouse cell lines. Addition of telomeric TTAGGG repeats was specific to telomeric oligonucleotide primers and sensitive to pretreatment with RNase A. In contrast to the hundreds of repeats synthesized by the human and Tetrahymena telomerase enzymes in vitro, mouse telomerase synthesized only one or two TTAGGG repeats onto telomeric primers. The products observed after elongation of primers with circularly permuted (TTAGGG)3 sequences and after chain termination with ddATP or ddTTP indicated that mouse telomerase pauses after the addition of the first dG residue in the sequence TTAGGG. The short length of the products synthesized by mouse telomerase was not due to a diffusible inhibitor in the mouse extract, because the human telomerase continued to synthesize long products when mixed with mouse fractions. Primer challenge experiments showed that the human enzyme synthesized long TTAGGG repeats processively in vitro, whereas the mouse telomerase appeared to be much less processive. The identification of short telomerase reaction products in mouse extracts suggests that extracts from other organisms may also generate only short products. This knowledge may aid in the identification of telomerase activity in organisms where activity has not yet been detected.
在几种不同的小鼠细胞系提取物中鉴定出了端粒酶活性。端粒TTAGGG重复序列的添加对端粒寡核苷酸引物具有特异性，并且对核糖核酸酶A预处理敏感。与人类和四膜虫端粒酶在体外合成的数百个重复序列不同，小鼠端粒酶仅在端粒引物上合成一两个TTAGGG重复序列。用环状排列的(TTAGGG)3序列延伸引物后以及用ddATP或ddTTP终止链后观察到的产物表明，小鼠端粒酶在添加序列TTAGGG中的第一个dG残基后会暂停。小鼠端粒酶合成的产物长度较短并非由于小鼠提取物中存在可扩散的抑制剂，因为当与人的组分混合时，人类端粒酶仍继续合成长产物。引物挑战实验表明，人类酶在体外能持续合成长的TTAGGG重复序列，而小鼠端粒酶的持续性似乎要低得多。在小鼠提取物中鉴定出短的端粒酶反应产物表明，来自其他生物体的提取物可能也只会产生短产物。这一知识可能有助于在尚未检测到活性的生物体中鉴定端粒酶活性。
