Lupski J R, Smiley B L, Blattner F R, Godson G N
Mol Gen Genet. 1982;185(1):120-8. doi: 10.1007/BF00333800.
A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A lambda phage library was constructed by ligating a Sau3A( decreases GATC) partial DNA digest of the entire E. coli chromosome into the lambda BamHI(G decreases GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRBl) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: lambda 3, lambda 4, lambda 5, lambda 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant lambda 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of lambda 4 using different restriction of fragments. Plasmids pGL444 and pBS10 5 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement of dnaGts chromosomal marker at nonpermissive 40 degrees C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.
已克隆并鉴定了大肠杆菌染色体上围绕dnaG基因的一个24千碱基对区域。通过将整个大肠杆菌染色体的Sau3A（切割GATC）部分DNA消化片段连接到λ噬菌体BamHI（切割GATCC）克隆载体Charon 28中，构建了一个λ噬菌体文库。进行部分消化以产生重叠的染色体片段，并允许沿着染色体进行步移。用含有rpoD基因的缺口平移质粒（pRRB1）对该文库进行探测，rpoD基因位于与dnaG相邻的66分钟处。从筛选的2500个噬菌斑中分离出四个与探针杂交的噬菌体：λ3、λ4、λ5、λ6。其中一个噬菌体重组体λ4被证明含有dnaG基因。通过使用不同的片段限制性内切酶对λ4进行亚克隆，构建了三个含有dnaG的重组质粒：pGL444、pGL445、pBS105。质粒pGL444和pBS105接受转座子Tn5诱变，并分离出88个插入到克隆区域的Tn5。通过对质粒进行限制性内切酶分析来定位Tn5插入的位置，并检查插入突变在40℃非允许温度下对dnaGts染色体标记的互补能力。通过这种方式确定了dnaG的相关物理图谱和遗传图谱。大量的Tn5插入定位到一个特定的900碱基对区域，我们认为该区域可能参与dnaG基因表达的调控。
