利用核定位信号筛选系统初步筛选小鼠胚胎核定位蛋白基因

在已建立的核定位信号 (nuclearlocalizationsignal,NLS)筛选系统的基础上 ,对这一系统进行了改进并对改进的系统进行了验证。将小鼠 1 1天胚胎cDNA文库插入改进后的筛选载体的多克隆位点 ,转化酵母宿主菌。然后将约 1 0 4 个酵母克隆接种于选择性平板上进行筛选 ,得到了 2 2个可在选择性培养基上生长的克隆。分析了其中 1 8个克隆的DNA序列 ,见到 1 3个克隆含有以正确读框融合的编码NLS的基因片段。取其中 3个克隆的插入片段与绿色荧光蛋白基因融合后在哺乳类细胞内表达 ,证明了其在哺乳类细胞中的核定位功能。研究证明 ,构建的核定位信号筛选系统 ,能够有效地从cDNA文库中筛选核定位蛋白的基因

Screening of Genes Encoding Nuclear Localization Signal in a Mouse Embryonic cDN A Library

A genetic screening system was established previou sl y for the cloning of gene fragments encoding nuclear localization signals (N LS). In the current study, the system was improved and its ability to distinguis h nuclear and cytoplasm localized proteins was verified by inserting a kn own NLS encoding gene. A mouse 10.5 d pc embryonic cDNA library was then inser ted into the cloning site of the screening vector and 22 out of 10 4 clones wer e shown to be positive after screening using the system. The cDNA inserts of som e of these clones were sequenced , and typical and untypical NLS were identif ied. The nuclear localizing activity of the cDNA inserts was further verified us ing the GFP fusion protein system. So, the system is useful in the cDNA library screening of genes encoding NLS bearing proteins.