Cimino-Reale G, Pascale E, Battiloro E, Starace G, Verna R, D'Ambrosio E
Istituto di Neurobiologia e Medicina Molecolare, CNR, Viale Marx 15-43, I-00137, Roma, Italy.
Nucleic Acids Res. 2001 Apr 1;29(7):E35. doi: 10.1093/nar/29.7.e35.
A typical G-rich telomeric DNA strand, which runs 5'-->3' toward the chromosome ends, protrudes by several nucleotides in lower eukaryotes. In human chromosomes long G-rich 3'-overhangs have been found. Apart from the standard G-rich tail, several non-canonical terminal structures have been proposed. However, the mechanism of long-tail formation, the presence and the role of these structures in telomere maintenance or shortening are not completely understood. In a search for a simple method to accurately measure the 3'-overhang we have established a protocol based on the ligation of telomeric oligonucleotide hybridized to non-denatured DNA under stringent conditions (oligonucleotide ligation assay with telomeric repeat oligonucleotide). This method enabled us to detect a large proportion of G-rich single-stranded telomeric DNA that was as short as 24 nt. Nevertheless, we showed G-tails longer than 400 nt. In all tested cells the lengths ranging from 108 to 270 nt represented only 37% of the whole molecule population, while 56-62% were <90 nt. Our protocol provides a simple and sensitive method for measuring the length of naturally occurring unpaired repeated DNA.
一条典型的富含鸟嘌呤的端粒DNA链，其朝着染色体末端的方向为5'→3'，在低等真核生物中会突出几个核苷酸。在人类染色体中已发现有长的富含鸟嘌呤的3'端悬突。除了标准的富含鸟嘌呤的尾巴外，还提出了几种非典型的末端结构。然而，长尾形成的机制、这些结构在端粒维持或缩短中的存在及作用尚未完全明了。在寻找一种准确测量3'端悬突的简单方法时，我们建立了一种基于在严格条件下将端粒寡核苷酸与非变性DNA杂交后进行连接的方案（端粒重复寡核苷酸的寡核苷酸连接测定）。该方法使我们能够检测到大量短至24个核苷酸的富含鸟嘌呤的单链端粒DNA。尽管如此，我们也发现了长度超过400个核苷酸的G尾。在所有测试细胞中，长度在108至270个核苷酸之间的分子仅占整个分子群体的37%，而56% - 62%的分子长度小于90个核苷酸。我们的方案提供了一种简单且灵敏的方法来测量天然存在的未配对重复DNA的长度。
