STRUCTURE AND GENETIC CONTROL OF COLICINES

这项建议的主旨是对人的遗传和生化分析 质粒DNA复制的调控和质粒的染色体外状态 细菌中的元素，特别强调细菌的性质和机制 所涉及的质粒特异性蛋白质和质粒核苷酸序列的作用 在受调控的质粒DNA复制的启动和分离中 细胞分裂过程中的质粒组分。一个相关的问题，遗传和 血浆不亲和性的生化机制(即，某些 在细菌细胞中稳定共存的质粒元素)也将受到强调。 努力将集中在R6K(多拷贝抗生素耐药性)质粒上 寄主范围窄的质粒)和RD2(每个细胞的有限拷贝数和宽拷贝数 主机范围)。将在体外和体内对大肠杆菌进行研究 Coli细胞和E.Coli提取物。在RK2的情况下，体内的质粒 复制和维护研究将扩展到遥远的细菌 与大肠杆菌有关。质粒不相容区域的分析 迷你F也将继续。最后，质粒Co1E1及其衍生物将 重组DNA技术在节理施工中的广泛应用 含R6K、RK2和mini-F及其衍生物的复制子在分析中的应用 复制的启动、质粒分离和调控机制 质粒不相容。对区域的广泛分析对 复制和不兼容属性将在以下级别执行 利用重组DNA技术重建质粒DNA，分离和鉴定 质粒复制突变体的特性，核苷酸序列分析， 参与调控的质粒特异性蛋白的特性 质粒复制和不相容。寄主范围广，质粒克隆 利用重组DNA进一步开发从RD2质粒衍生的载体 程序。最后，将努力发展自主环状DNA 组织培养中生长的动物细胞中的复制子。

The major thrust of this proposal is a genetic and biochemical analysis of the regulation of plasmid DNA replication and the extrachromosomal state of plasmid elements in bacteria with particular emphasis on the nature and mechanism of action of plasmid specified proteins and plasmid nucleotide sequences involved in the regulated initiation of plasmid DNA replication and the segregation of plasmid elements during cell division. A related problem, the genetic and biochemical mechanisms of plasmic incompatibility (i.e., the failure of certain plasmid elements to co-exist stably in a bacterial cell) also will be stressed. Efforts will be concentrated on plasmids R6K (multi-copy antibiotic resistance plasmid of narrow host range) and RD2 (limited copy number per cell and broad host range). Studies will be carried out in vitro and in vivo with Escherichia coli cells and E. coli extracts. In the case of RK2 the in vivo plasmid replication and maintenance studies will be extended to bacteria distantly related to E. coli. An analysis of the incompatibility regions of plasmid mini-F also will be continued. Finally, plasmid Co1E1 and its derivatives will be used extensively in the construction by recombinant DNA techniques of joint replicons with R6K, RK2 and mini-F and their derivatives in the analysis of regulatory mechanisms for initiation of replication, segregation of plasmids and plasmid incompatibility. Extensive analyses of regions essential for replication and incompatibility properties will be carried out at the levels of plasmid DNA reconstructions using recombinant DNA techniques, isolation and characterization of plasmid replication mutants, nucleotide sequence analysis, characterization of plasmid specified proteins involved in the regulation of plasmid replication and incompatability. A broad host range, plasmid cloning vehicle derived from plasmid RD2 will be further developed using recombinant DNA procedures. Finally, efforts will be made to develop autonomous circular DNA replicons in animal cells grown in tissue culture.