Skowron P M, Swaminathan N, McMaster K, George D, Van Etten J L, Mead D A
Molecular Biology Resources Inc., Milwaukee, WI 53210, USA.
Gene. 1995 May 19;157(1-2):37-41. doi: 10.1016/0378-1119(94)00564-9.
The gene (cviJIR) encoding the two/three-base R.CviJI eukaryotic restriction endonuclease (ENase) from IL-3A virus-infected Chlorella was cloned into Escherichia coli. A high frequency of DNA cleavage by R.CviJI required overexpression of the gene encoding the M.CviJI methyltransferase prior to cloning the gene for the ENase. Both genes were sequenced and their organization was determined to be in head-to-tail order. The open reading frame coding for R.CviJI can potentially translate a 41.4-kDa protein; however, in the E. coli host, a truncated version of the enzyme is produced (32.5 kDa). The recombinant ENase does not exhibit ATP-induced 'star' activity (R.CviJI cleaves at RGCY, while R.CviJI* also cleaves at RGCR and YGCY, but not at YGCR), as is characteristic for native R.CviJI. The very high frequency of DNA cleavage by R.CviJI* was exploited in the development of a quasi-random shotgun library method. R.CviJI*-generated oligodeoxyribonucleotides were applied to improve certain molecular biology applications, i.e., DNA labeling, detection, high-resolution restriction mapping, amplification and epitope mapping.
将来自感染IL - 3A病毒的小球藻的编码两/三碱基R.CviJI真核限制性内切酶（ENase）的基因（cviJIR）克隆到大肠杆菌中。在克隆ENase基因之前，R.CviJI对DNA的高效切割需要先过表达编码M.CviJI甲基转移酶的基因。对这两个基因进行了测序，并确定它们的排列顺序是头对头的。编码R.CviJI的开放阅读框可能翻译出一个41.4 kDa的蛋白质；然而，在大肠杆菌宿主中，产生的是该酶的截短版本（32.5 kDa）。重组ENase不表现出ATP诱导的“星号”活性（R.CviJI在RGCY处切割，而R.CviJI也在RGCR和YGCY处切割，但不在YGCR处切割），这是天然R.CviJI的特征。R.CviJI对DNA的高效切割被用于开发一种准随机鸟枪法文库方法。R.CviJI*产生的寡脱氧核糖核苷酸被用于改进某些分子生物学应用，即DNA标记、检测、高分辨率限制性图谱绘制、扩增和表位作图。
