Hansen B S, Manning P A, Achtman M
J Bacteriol. 1982 Apr;150(1):89-99. doi: 10.1128/jb.150.1.89-99.1982.
The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.
利用源自R100的小质粒，主要是质粒pSM6，对F类质粒R100的tra（转移）操纵子的远端区域进行了研究。转座子Tn5（赋予卡那霉素抗性）被插入到pSM6的不同位置，并对转座衍生物进行测试，以检测其对F性因子特定tra突变体的互补能力。由此，tra基因traH、G、T和D被定位在质粒R100上。构建了pSM6的限制性图谱，并通过对质粒DNA进行限制性内切酶消化以及利用几个限制性位点位于转座子反向重复区域这一事实，对插入位点进行了定位。通过对携带各种插入质粒的小细胞中合成的蛋白质进行放射性标记，随后进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，鉴定了traG、S、T和D基因的产物。从这些数据推断出另一个转移基因traI的存在。还鉴定了另一种蛋白质，即r2 - A蛋白，并对其基因进行了定位。基于这些数据，构建了R100的tra操纵子该区域的最佳拟合物理图谱。结果证实，远端转移基因的总体顺序和大小与F性因子中的相同，但表明在所有基因产物方面存在差异。根据转移区域的遗传和物理同源性（Manning等人，《细菌学杂志》150:76 - 88）讨论了这些差异的意义。
