Calhoun D H, Traub L, Wallen J W, Gray J E, Guterman S K
Mol Gen Genet. 1984;193(2):205-9. doi: 10.1007/BF00330668.
The location of the rho gene and its position relative to the ilv genes of Escherichia coli K-12 was analyzed using genetic criteria, restriction enzyme cleavage, and maxicell analysis. Plasmids were constructed with deletions of the rho gene introduced in vitro, and lambda ilv-gal derivatives of lambda ilv-rho bacteriophage were isolated by recombination in vivo. A HindIII restriction fragment of 8 kilobases (kb) previously shown to contain at least part of the rho gene (Gray et al. 1981) was cloned into plasmid pMC81. This vector has transcription stop sites that present read-through expression of cloned genes from either direction, and cloning sites upstream of the lacZ gene coding for beta-galactosidase. The position of the rho gene and flanking sequences required for its expression were further localized to a region of approximately 2 kb by introducing deletions using restriction enzyme treatment of these plasmids. A promoter in the rho region was found to direct beta-galactosidase synthesis in these plasmid derivatives. Derivatives of lambda ilv-rho phage were isolated in vivo by pyrophosphate chelation selection for phage with reduced genome size. Restriction enzyme analysis of twelve of these derivatives revealed an unexpected bias towards phage recombinants as opposed to simple internal deletions.
利用遗传学标准、限制性内切酶切割和大细胞分析，对大肠杆菌K-12的rho基因位置及其相对于ilv基因的位置进行了分析。构建了体外导入rho基因缺失的质粒，并通过体内重组分离出lambda ilv-rho噬菌体的lambda ilv-gal衍生物。先前已证明包含至少部分rho基因的一个8千碱基（kb）的HindIII限制性片段（Gray等人，1981年）被克隆到质粒pMC81中。该载体具有转录终止位点，可阻止从任一方向对克隆基因的通读表达，并且在编码β-半乳糖苷酶的lacZ基因上游有克隆位点。通过对这些质粒进行限制性酶切处理引入缺失，rho基因的位置及其表达所需的侧翼序列进一步定位到大约2 kb的区域。在这些质粒衍生物中发现rho区域的一个启动子可指导β-半乳糖苷酶的合成。通过焦磷酸螯合选择基因组大小减小的噬菌体，在体内分离出lambda ilv-rho噬菌体的衍生物。对其中12种衍生物的限制性酶切分析显示，与简单的内部缺失相反，噬菌体重组体存在意外的偏向性。
