CONTROL OF PLASMID R1162 DNA REPLICATION

一个控制DNA复制的模型的主要特征 将测试宽宿主范围质粒R1162：（1） 直接重复序列的R1162 DNA结合质粒编码的蛋白质？ 这 序列将被化学合成并测试特异性结合 纯化的质粒蛋白质，不相容性的表达和抑制， R1162 DNA体外复制。 将识别重要的碱基对 从影响这些特性的突变的位置， 在DNA酶I和甲基化试剂存在下的碱基保护。 （二） DNA合成开始于反向重复序列吗？ 的重要性 反向重复DNA的复制，以及DNA合成的位置 将通过表征oriV突变来确定， 假回复突变体，并通过映射新生的DNA片段分离， 复制分子 (3)质粒蛋白会沿着DNA吗？ 蛋白质-DNA复合物将被UV照射捕获并被标记。 胶体金抗体 蛋白质沿着质粒DNA的分布 将分析后，这些配合物的检查电子 显微镜 (4)如何控制RepI区域的表达？ 的 两个物种中RepI mRNA的起始点将通过S1 映射. 启动子内的关键碱基对将从 分离和定位“上调启动子”突变。 放射性 杂交探针将用于检测推定的反转录物，和 其在调节中的作用通过测量这种RNA的水平来确定 在含有铜质粒的细胞中。 施加控制的水平 将从基因融合的行为中得出结论，并通过定量 在存在和不存在蛋白质的情况下对RepI mRNA的量进行估计， 负调节器

Principal features of a model for the control of DNA replication of the broad host-range plasmid R1162 will be tested: (1) Does the directly-repeated sequence of R1162 DNA bind plasmid-encoded protein? This sequence will be chemically synthesized and tested for specific binding of purified plasmid proteins, expression of incompatibility and inhibition of R1162 DNA replication in vitro. Significant base-pairs will be identified from the locations of mutations affecting these properties, and by studies of base protection in the presence of DNaseI and methylating agents. (2) Does DNA synthesis begin within the inverted repeat? The importance of the inverted repeat DNA for replication, and the positions where DNA synthesis is initiated, will be determined by characterizing oriV-mutations and pseudorevertants, and by mapping nascent DNA fragments isolated from replicating molecules. (3) Does a plasmid protein track along the DNA? Protein-DNA complexes will be trapped by UV irradiation and labelled by colloidal gold-antibody. The distribution of protein along the plasmid DNA will be analyzed following the examination of these complexes by electron microscopy. (4) How is expression of the RepI region controlled? The starting point of RepI mRNA in two species will be determined by S1 mapping. Key base-pairs within the promoter will be identified from the isolation and mapping of "up-promoter" mutations. A radioactive hybridizing probe will be used to detect a putative countertranscript, and its role in regulation determined by the measurement of levels of this RNA in cells containing cop plasmids. The level at which control is exerted will be concluded from the behavior of gene fusions, and by quantitative estimates of the amounts of RepI mRNA made in the presence and absence of a negative regulator.