Chevallier M R
Mol Cell Biol. 1982 Aug;2(8):977-84. doi: 10.1128/mcb.2.8.977-984.1982.
The uracil permease gene of the yeast Saccharomyces cerevisiae was cloned on a hybrid plasmid which replicates autonomously in both yeast and Escherichia coli. Cloning was carried out by complementation in yeast. The smallest DNA fragment found to complement the uracil permease deficiency in recipient yeast cells measured approximately 2.3 kilobases. In strains transformed by the plasmid with the uracil permease gene inserted, initial rates of uracil uptake increased up to 25 times more than the rates found in the wild type. Using DNA probes carrying several regions of the cloned gene, I showed that a strain carrying the dhul-I mutation, which is not linked to the permease structural gene and is responsible for enhanced uptake velocity of uracil, had enhanced transcription of the permease gene. By using DNA probes recloned in phage M13 mp7, the direction of transcription of the permease gene relative to the restriction map was deduced. A half-life of 2 min was found for the permease mRNA in labeling kinetics experiments.
酿酒酵母的尿嘧啶通透酶基因被克隆到一个能在酵母和大肠杆菌中自主复制的杂种质粒上。克隆是通过酵母中的互补作用进行的。发现能互补受体酵母细胞中尿嘧啶通透酶缺陷的最小DNA片段约为2.3千碱基。在插入了尿嘧啶通透酶基因的质粒转化的菌株中，尿嘧啶摄取的初始速率比野生型高出多达25倍。使用携带克隆基因几个区域的DNA探针，我发现携带dhul-I突变的菌株（该突变与通透酶结构基因不连锁且导致尿嘧啶摄取速度增强），其通透酶基因的转录增强。通过使用在噬菌体M13 mp7中重新克隆的DNA探针，推断出通透酶基因相对于限制酶切图谱的转录方向。在标记动力学实验中发现通透酶mRNA的半衰期为2分钟。
