CONTROL OF PLASMID R1162 DNA REPLICATION

细胞DNA复制控制模型的主要特征 将测试寄主范围广泛的质粒R1162：(1) R1162 DNA的直接重复序列结合了质粒编码的蛋白？这 序列将通过化学合成并进行特定结合测试 纯化的质粒蛋白，不相容的表达和抑制 R1162DNA体外复制。重要的碱基对将被确定 根据影响这些特性的突变的位置，以及研究 在DNaseI和甲基化试剂存在的情况下进行碱基保护。(2) DNA合成是从反向重复开始的吗？重要的一点是 用于复制的反向重复DNA，以及DNA合成的位置 将通过表征oriV突变和 通过对分离的新的DNA片段进行图谱分析， 复制分子。(3)质粒蛋白沿着DNA追踪吗？ 蛋白质-DNA复合体将被紫外光捕获并被标记 胶体金-抗体。蛋白质沿质粒DNA的分布 将在对这些络合物进行电子检查后进行分析 显微镜。(4)REPI区域的表达如何调控？这个 两个物种中REPI基因的起点将由S1决定 映射。启动子内的关键碱基对将从 “上启动子”突变的分离和定位。一种放射性物质 杂交探针将用于检测假定的反转录本，以及 它在调节中的作用由这种RNA水平的测量确定 在含有COP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，并通过量化 在存在和不存在A的情况下对REPI信使核糖核酸数量的估计 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.