CONJUGATIVE MOBILIZATION OF PLASMID R1162

广宿主范围质粒R1162的接合动员 需要oriT，一个顺式活性的，38个碱基对的位点， 至少四种质粒mob基因的产物。 突变oriT DNA将被测试，以确定重要的碱基对， 移动、缀合依赖性重组和切口 放松复合体。 此外，重组的位点， 而oriT内的弛豫络合物诱导的切口将是 映射。 MOB基因产物将通过检测 在含有克隆片段的微细胞中制备的多肽 R1162 DNA;然后将相应的开放阅读框 通过DNA碱基测序定位。 方向和搁浅 将确定动员期间质粒DNA转移的 通过杂交新转移的DNA，分离自微细胞， 用特定的DNA探针 暴民基因的突变 与oriT相互作用的产物将作为假回复突变体获得 不可移动的oriT质粒。 变异的暴民基因 通过互补和重组绘制。 最后 在松弛复合物切口位点共价连接的多肽 将被分离，并且氨基酸序列部分 测定 然后将该序列与碱基进行比较， mob基因的序列，以确定基因编码 多肽。

Conjugative mobilization of broad host-range plasmid R1162 requires both oriT, a cis-active, 38 base-pair site, and the products of at least four plasmid mob genes. Mutagenized oriT DNA will be tested to identify the base-pairs important for mobilization, conjugation-dependent recombination, and nicking by relaxation complex. In addition, the sites of recombination, and relaxation complex-induced nicking within oriT will be mapped. The mob gene products will be identified by examining the polypeptides made in minicells containing cloned fragments R1162 DNA; the corresponding open reading frames will then be located by DNA base sequencing. The direction and strandedness of plasmid DNA transfer during mobilization will be determined by hybridizing newly-transferred DNA, isolated from minicells, with specific DNA probes. Mutations in mob genes whose products interact with oriT will be obtained as pseudorevertants of nonmobilizable, oriT-plasmids. The altered mob genes will be mapped by complementation and recombination. Finally, the polypeptide covalently joined at the relaxation complex nick site will be isolated, and the amino acid sequence partially determined. This sequence will then be compared to the base sequence of the mob genes, in order to identify the gene encoding the polypeptide.