STRUCTURE AND GENETIC CONTROL OF COLICINES

这项提案中的研究涉及到基因的特征 以及负责调控复制的生化机制 并且为了稳定地维持广宿主范围的RK2在 细菌。这个质粒会对抗生素产生抗药性 四环素、氨苄西林和卡那霉素，在 在广泛的革兰氏阴性菌中处于染色体外状态。二 该质粒组分是其绝对必需的 复制是编码两个复制起始的trfA基因 蛋白质(44 kDa和33 kDa)，其中较小的是由于 在同一个开放阅读框架中的内部翻译开始，以及 复制起始序列，其主要特征为8个17个碱基对 以五个和三个为一组重复(迭代)。44 kDa和33 kDa 在细菌中，蛋白质的活性基本相同。 大肠埃希氏菌和这两种蛋白都与迭代序列在 RK2复制源。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(S)和复制起始处的序列，它们是 负责管理复制的启动和 该抗生素耐药质粒在大范围内维持的能力 细菌的范围。这些研究还将关注对以下方面的分析 TrfA蛋白/起始序列复合体与TfA的相互作用 从几种革兰氏阴性菌中分离出特异性宿主复制蛋白 细菌。这些分析将在体内和体外进行，并将 涉及野生型和突变型TrfA蛋白，包括缺陷和 复制变种人。将尝试确定TrfA的区域 负责其与复制的各种相互作用的蛋白质 来源和宿主复制蛋白。最后，RK2的一个区域 一种似乎编码广泛宿主范围的质粒区的质粒 为了理解这种机制，将对其功能进行考察(S) 负责将一个质粒分割成细胞上的子细胞 组织。除了提供有关监管的信息外 启动复制和稳定维持一个 细菌中的染色体外成分，这一基本成分的实际影响 研究将建筑的高拷贝数量和稳定保持 广泛应用于革兰氏阴性杆菌基因克隆的质粒载体 具有医学和农业重要性的细菌。

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.