Green T L, Hart R P
Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102.
Mol Cell Biol. 1988 Apr;8(4):1839-41. doi: 10.1128/mcb.8.4.1839-1841.1988.
Previous studies have shown that a sequence element downstream of the poly(A) addition site is required for efficient cleavage in vivo. We tested a group of downstream element point mutations in an in vitro reaction using HeLa cell nuclear extract as a source of cleavage activity. In close agreement with earlier studies (M. A. McDevitt, R. P. Hart, W. W. Wong, and J. R. Nevins, EMBO J. 5:2907-2913, 1986), a downstream element from the adenovirus E2a gene directed a higher level of cleavage activity than one from the simian virus 40 early gene. Furthermore, a single-base change in the downstream element could result in a decrease in cleavage activity of about 50-fold. That these mutations have similar effects in vivo and in vitro indicates that the HeLa cell nuclear extract system contains all of the factors required to study the mechanism of sequence recognition.
先前的研究表明，聚腺苷酸（poly(A)）添加位点下游的一个序列元件是体内有效切割所必需的。我们在体外反应中使用HeLa细胞核提取物作为切割活性来源，测试了一组下游元件点突变。与早期研究（M. A. McDevitt、R. P. Hart、W. W. Wong和J. R. Nevins，《欧洲分子生物学组织杂志》5:2907 - 2913，1986年）密切一致的是，腺病毒E2a基因的下游元件比猴病毒40早期基因的下游元件指导更高水平的切割活性。此外，下游元件中的单个碱基变化可能导致切割活性降低约50倍。这些突变在体内和体外具有相似的作用，这表明HeLa细胞核提取物系统包含研究序列识别机制所需的所有因子。
