Hertzberg K M, Gemmill R, Jones J, Calvo J M
Gene. 1980 Jan;8(2):135-52. doi: 10.1016/0378-1119(80)90033-5.
Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.
用经EcoRI处理的鼠伤寒沙门氏菌DNA与质粒pSC101 DNA的连接混合物将大肠杆菌亮氨酸营养缺陷型转化为原养型后，分离得到了携带鼠伤寒沙门氏菌部分亮氨酸操纵子的重组质粒。质粒pCV11和pCV13含有与载体连接的3.4×10⁶道尔顿的DNA片段，其亮氨酸操纵子方向相反。确定了亮氨酸操纵子相对于质粒基因的方向。将3.4×10⁶道尔顿的片段连接到质粒pMB9的EcoRI位点，得到质粒pCV12（方向与pCV11相同）和pCV14（方向与pCV13相同）。酶活性测定和互补试验结果表明，这些质粒携带功能性的leuA、leuB和leuC基因，但不携带功能性的leuD基因。此外，以下结果表明它们具有功能性的亮氨酸控制区和启动子。在亮氨酸限制条件下，质粒亮氨酸基因的表达明显增强，而将亮氨酸启动子突变引入相对于质粒基因呈任一方向排列的操纵子中，对亮氨酸操纵子的表达有强烈的负面影响。与在亮氨酸启动子起始的转录相比，来自质粒启动子的转录通读（如果确实发生的话）一定很少。从在阻遏和去阻遏条件下生长的亮氨酸营养缺陷型以及由于leuO、leuS和flrB突变而使亮氨酸操纵子去阻遏的原养型菌株中分离RNA。通过与质粒pCV12 DNA杂交测定的亮氨酸mRNA合成速率，在每种情况下都与亮氨酸酶水平成正比。
