Johnson L M, Snyder M, Chang L M, Davis R W, Campbell J L
Cell. 1985 Nov;43(1):369-77. doi: 10.1016/0092-8674(85)90042-x.
A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants.
利用在λgt11载体中构建的酵母基因组DNA表达文库，以抗酵母DNA聚合酶I的抗体筛选，从而分离出编码DNA聚合酶I的基因。通过几个标准确定了DNA聚合酶I基因的身份。首先，克隆编码的蛋白质与DNA聚合酶I存在免疫相关性。其次，含有克隆于高拷贝数质粒YEp24中的该基因的细胞，其聚合酶活性在酵母提取物中检测时会过量产生4至5倍。最后，将该基因插入噬菌体T7启动子下游，可在大肠杆菌中合成酵母DNA聚合酶I。基因破坏和Southern杂交实验表明，该聚合酶由一个必需的单拷贝基因编码。对含有破坏基因的萌发孢子进行检查，发现其存在核分裂缺陷以及复制突变体典型的终末表型。
