Yoakum G H, Yeung A T, Mattes W B, Grossman L
Proc Natl Acad Sci U S A. 1982 Mar;79(6):1766-70. doi: 10.1073/pnas.79.6.1766.
We have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under pL control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, we developed a genetic selection using the basal level of expression (30 degrees C) from pL in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-UV-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under pL control rapidly. This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (i) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (ii) production of high-level amplification for the gene product of interest by screening for post-UV-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids. The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli.
我们构建了一种杂交pKC30-uvrA质粒（pGHY5003），其中uvrA基因的转录可在pL控制下被诱导，从而将uvrA基因产物扩增至细胞蛋白质的7%。为构建pGHY5003，我们利用热敏性cI857溶原菌中pL的基础表达水平（30℃）进行遗传筛选，以便从含有多种产物的重组DNA混合物中分离出插入到pKC30的Hpa I位点的经过适当改造的修复基因。此外，一种紫外线照射后的放射性标记方法被用于快速筛选由pL控制转录所指导的温度诱导型多肽合成的插入片段。这对于分离插入到质粒pKC30的Hpa I位点且具有以下特征的基因应具有普遍用途：（i）从大量连接到pKC30的Hpa I位点的核酸外切酶改造片段群体中筛选出具有遗传功能的杂交质粒，以及（ii）通过筛选杂交质粒合成的多肽的紫外线照射后温度诱导性，来实现对感兴趣基因产物的高水平扩增。从pGHY5003获得的uvrA基因产物的扩增水平比对数期大肠杆菌中uvrA蛋白水平的估计值高约10000倍。
