STRUCTURE AND GENETIC CONTROL OF COLICINES

这项提案中的研究涉及遗传学的表征， 以及负责复制调节的生化机制 并且为了使广宿主范围质粒RK 2在 细菌 这种质粒能对抗生素产生抗药性 四环素，氨苄青霉素和卡那霉素，并稳定地保持在 在广泛的革兰氏阴性细菌中的染色体外状态。 两 这种质粒的组成部分是绝对需要的， trfA基因编码两个复制起始点， 蛋白质（44 kDa和33 kDa），其中较小的是一个结果， 在同一开放阅读框架中的内部平移开始，以及 复制起点序列，其主要特征是含有8个17 bp 以五个和三个为一组的重复（iterons）。 44 kDa和33 kDa 蛋白质在细菌中的活性基本相等 大肠杆菌和这两种蛋白质特异性地结合在 RK 2复制起点。 这项建议的主旨是 检查TrfA复制之间相互作用的性质， 蛋白质和复制起点处的序列， 负责调节复制的起始和 这种抗生素抗性质粒能够在广泛的 细菌的范围。 这些研究还将涉及分析 TrfA蛋白/起始序列复合物与 从几种革兰氏阴性菌中分离的特异性宿主复制蛋白 细菌 将在体内和体外进行分析， 涉及野生型和突变型TrfA蛋白，包括缺陷型和 复制突变体将尝试鉴定TrfA的区域， 负责与复制的各种相互作用的蛋白质 起源和宿主复制蛋白。 最后，RK 2的一个区域 似乎编码宽宿主范围质粒分配的质粒 将检查功能，以了解机制 负责将质粒分配到子细胞， 师. 除了提供关于监管的信息外， 启动复制和稳定维持一个 细菌中的染色体外元素，这一基本原理的实际后果 研究将构建高拷贝数并稳定保持 质粒载体用于广泛的革兰氏阴性菌中的基因克隆 在医学和农业上有重要意义的细菌。

The research in this proposal deals with the characterization of genetic and biochemical mechanisms responsible for the regulation of replication and for stable maintenance of the broad-host-range plasmid RK2 in bacteria. This plasmid specifies resistance to the antibiotics tetracycline, ampicillin and kanamycin and is stably maintained in the extrachromosomal state in a wide range of Gram-negative bacteria. Two components of this plasmid that are absolutely required for its replication are the trfA gene that encodes two replication initiation proteins (44 kDa and 33 kDa), the smaller of which is the result of an internal translational start in the same open reading frame, and a replication origin sequence that contains as its main feature eight 17 bp repeats (iterons) in groups of five and three. The 44 kDa and 33 kDa proteins are essentially equivalent in activity in the bacterium Escherichia coli and both proteins specifically bind to the iterons at the RK2 replication origin. The major thrust of this proposal is to examine the nature of the interactions between the TrfA replication protein(s) and the sequence at the replication origin that are responsible for the regulation of initiation of replication and the ability of this antibiotic resistance plasmid to be maintained in a wide range of bacteria. These studies also will be concerned with analyses of the interactions between the TrfA protein/origin sequence complex and specific host replication proteins isolated from several Gram-negative bacteria. The analyses will be carried out in vivo and in vitro and will involve both wild-type and mutant TrfA proteins, including defective and copy-up mutants. An attempt will be made to identify regions of the TrfA protein responsible for its various interactions with the replication origin and the host replication proteins. Finally, a region of the RK2 plasmid that appears to encode a broad-host-range plasmid partitioning function will be examined in order to understand the mechanism(s) responsible for the partitioning of a plasmid to daughter cells upon cell division. In addition to providing information on the regulation of initiation of replication and the stable maintenance of an extrachromosomal element in bacteria, a practical fallout of this basic study will be the construction of high-copy number and stably maintained plasmid vectors for gene cloning in a wide range of Gram-negative bacteria of medical and agricultural importance.