CONTROL OF PLASMID R1162 DNA REPLICATION

质粒 R1162 DNA 复制的控制

• 批准号: 3291361
• 负责人: RICHARD J MEYER
• 金额: $ 13.48万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291361
• 关键词: DNA binding protein; Escherichia coli; bacterial virus; binding proteins; electron microscopy; gene expression; genetic manipulation; genetic mapping; genetic models; genetic promoter element; genetic recombination; laboratory rabbit; messenger RNA; model design /development; molecular cloning; mutant; nucleic acid hybridization; nucleic acid sequence; plasmids; radiation genetics; radiotracer; ultraviolet radiation

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.

控制DNA复制的模型的主要特征 将测试广泛的宿主范围R1162：（1） R1162 DNA的直接重复序列结合质粒编码的蛋白？ 这 序列将被化学合成，并测试以特异性结合 纯化的质粒蛋白，表达不兼容和抑制 R1162 DNA在体外复制。 将确定重要的碱基对 从影响这些特性的突变的位置，并通过研究 在存在DNASEI和甲基化剂的情况下进行基础保护。 （2） DNA合成是否在反向重复范围内开始？ 重要性 倒重复DNA复制，以及DNA合成的位置 启动，将通过表征Oriv-Mutations和 伪逆想者，并通过绘制从中分离出的新生DNA片段 复制分子。 （3）质粒蛋白会沿DNA轨道吗？ 蛋白质-DNA复合物将被紫外线照射捕获，并被标记 胶体金抗体。 沿质粒DNA的蛋白质分布 电子将在检查这些复合物后分析 显微镜。 （4）如何控制REPI区域的表达？ 这 在两个物种中的repi mRNA的起点将由S1确定 映射。 将从 隔离和映射“上启动器”突变。 放射性 杂交探针将用于检测推定的反转录，并 它在由该RNA水平的测量中确定的调节中的作用 在含有COP质粒的细胞中。 控制控制的水平 将从基因融合的行为和定量中得出结论 在存在和不存在的情况下估计的repi mRNA量 负调节器。