Miyajima A, Nakayama N, Miyajima I, Arai N, Okayama H, Arai K
Nucleic Acids Res. 1984 Aug 24;12(16):6397-414. doi: 10.1093/nar/12.16.6397.
A cDNA cloning vector that allows expression in Saccharomyces cerevisiae has been developed using the plasmid primer approach described by Okayama and Berg [Mol. Cell. Biol. 2:161-170(1982)]. The vector contains ARS1 and TRP1 for plasmid maintenance in yeast and the ADC1 or GAL1 promoter and the TRP5 terminator for expression of the cloned cDNA. Using this system, several recombinants with nearly full-length GAL1 cDNA inserts in a cDNA library made with galactose-induced yeast mRNA were identified. By measurement of galactokinase mRNA and its protein, the expression of GAL1 cDNA was shown to be under the control of the promoter placed upstream of the cDNA insert. Nucleotide sequence analysis revealed that the 3'-ends of the GAL1 cDNA inserts were not unique, indicating that polyA tails were added to GAL1 transcripts at multiple sites in the GAL1 gene. Genetic complementation of appropriate yeast mutants permitted the isolation of clones containing the coding sequences for GAL1, HIS3, and LEU2 from the same cDNA library.
利用冈山县和伯格所描述的质粒引物方法[《分子细胞生物学》2:161 - 170(1982)]，开发了一种能在酿酒酵母中表达的cDNA克隆载体。该载体含有用于在酵母中维持质粒的ARS1和TRP1，以及用于克隆cDNA表达的ADC1或GAL1启动子和TRP5终止子。使用这个系统，在一个用半乳糖诱导的酵母mRNA构建的cDNA文库中，鉴定出了几个带有几乎全长GAL1 cDNA插入片段的重组体。通过测量半乳糖激酶mRNA及其蛋白质，表明GAL1 cDNA的表达受位于cDNA插入片段上游的启动子控制。核苷酸序列分析显示，GAL1 cDNA插入片段的3'端并非唯一，这表明在GAL1基因的多个位点上，GAL1转录本都添加了聚腺苷酸尾巴。对合适的酵母突变体进行遗传互补，使得能够从同一个cDNA文库中分离出含有GAL1、HIS3和LEU2编码序列的克隆。
