Kawamura M, Takagi M, Yano K
Gene. 1983 Oct;24(2-3):157-62. doi: 10.1016/0378-1119(83)90075-6.
Gene libraries of DNA from an n-alkane-assimilating yeast strain, Candida maltosa IAM12247, were constructed, using Escherichia coli plasmid vector pBR322. A LEU gene from C. maltosa was cloned, and found to complement leu- mutations in E. coli and Saccharomyces cerevisiae. In E. coli, the LEU gene in the cloned yeast DNA fragment was efficiently expressed when inserted into the vector in one orientation, while in the other orientation, it was expressed only weakly. In S. cerevisiae, the Candida LEU gene was efficiently expressed when inserted into a shuttle vector pRC3 in both orientations, suggesting that the isolated Candida DNA fragment contains a promoter sequence of Candida in front of the LEU gene, which is operative in S. cerevisiae but not in E. coli. In addition, our data suggest that the cloned LEU fragment also contains an ARS (autonomously replicating sequence) site of C. maltosa.
利用大肠杆菌质粒载体pBR322构建了来自正烷烃同化酵母菌株麦芽糖假丝酵母IAM12247的DNA基因文库。克隆了麦芽糖假丝酵母的一个亮氨酸基因（LEU基因），发现它能互补大肠杆菌和酿酒酵母中的亮氨酸突变。在大肠杆菌中，克隆的酵母DNA片段中的LEU基因以一种方向插入载体时能高效表达，而以另一种方向插入时则表达较弱。在酿酒酵母中，当将假丝酵母的LEU基因以两种方向插入穿梭载体pRC3时都能高效表达，这表明分离的假丝酵母DNA片段在LEU基因前含有假丝酵母的启动子序列，该序列在酿酒酵母中起作用但在大肠杆菌中不起作用。此外，我们的数据表明克隆的LEU片段还含有麦芽糖假丝酵母的一个自主复制序列（ARS）位点。
