GENETIC CONTROL OF PHAGE T4 DNA REPLICATION

这项工作的总体目标是了解基因 调控复制DNA合成的机制 噬菌体T4聚合酶及其与其他细菌的相互作用 T4DNA复制复合体的蛋白质。特别的 我们感兴趣的是聚合酶调节的机制 它自己在体内合成。我们想知道这项规定是否 发生在转录或翻译水平上。另外，我们计划 融合结构基因的不同NH2末端片段 T4 DNA聚合酶(T4基因43)与大肠杆菌LacZ(β- 半乳糖苷酶)基因对嵌合体合成的影响 具有与不同N2H-相关的活性的蛋白质- 单一多肽(896个氨基酸)T4的末端长度 基因43产物。一种无细胞转录/翻译偶联 检测系统和纯化的转录和翻译检测将 被用来重建自身抑制的机制 T4DNA聚合酶的体外扩增及靶位定位 (运算符)用于镇压。DNA聚合酶附件的作用 控制基因43表达的蛋白质也将在 这些都在维托分析中。拟议的研究可能会揭示T4 DNA聚合酶的生物合成受 其他复制蛋白的可用性(对 复制复杂性)。一旦基因43的目标(操作者) 自体抑制物已被鉴定，它将在体外被改变 识别重要的核苷酸残基的操作 T4DNA聚合酶对操作者的识别 辅抑制物。43-基因合成的重组蛋白 LacZ融合将被表征为体内特性和 核酸结合抑制物的提纯和检测 体外活性。会对不同的人产生抗体 T4 DNA聚合酶的蛋白质片段，用于 突变体内在活性的纯化和分析 多肽。

The overall objective of this work is to understand the genetic mechanisms that regulate synthesis of the replication DNA polymerase of bacteriophage T4 and its interactions with other proteins of the T4 DNA replication complex. Of particular interest to us is the mechanism by which the polymerase regulates its own synthesis in vivo. We want to find out if this regulation occurs at the transcriptional or translational levels. Also, we plan to fuse different NH2-terminal segments of the structural gene for T4 DNA polymerase (T4 gene 43) to an E. coli lacZ (beta- galactosidase) gene in order to effect the synthesis of chimeric proteins that harbor activities associated with different N2H- terminal lengths of the single polypeptide (896 amino acid) T4 gene 43 product. A coupled transcription/translation cell-free assay system and purified transcription and translation assays will be used to reconstruct the mechanism of autogenous repression by T4 DNA polymerase in vitro and to localize the target site (operator) for repression. The roles of DNA polymerase accessory proteins in control of gene 43 expression will also be tested in these in vitor assays. The proposed studies may reveal that T4 DNA polymerase biosynthesis is coordinately controlled by availability of other replication proteins (global regulation of the replication complex). Once the target (operator) for gene 43 autogenous repressor is identified, it will be altered by in vitro manipulations in order to identify nucleotide residues important in recognition of the operator by T4 DNA polymerase and added corepressors. The recombinant proteins synthesized by gene 43- lacZ fusions will be characterized for in vivo properties and purified and assayed for nucleic-acid-binding and repressor activities in vitro. Antibodies will be generated to different protein segments of the T4 DNA polymerase and used for purification of and analysis of activities intrinsic to mutant peptides.