REGULATION OF EXOGENOUSLY INDUCED OPERON

沙门氏菌磷酸甘油酸转运系统的表达 小鼠伤寒杆菌是由外源诱导剂诱导的，而不是内源诱导剂诱导的。我们有 以pBR322为载体，将pGT系统克隆到大肠杆菌中。这个 克隆的DNA包含自克隆系统以来所需的所有调控元件 与鼠伤寒沙门氏菌的调控方式相同。由此开展的前期工作 实验室表明操纵子是负控制的 存在抑制物和激活剂，它们可以抵消和中和这种作用 抑制者。因为外源基因诱导的新颖性 表达，监管机制将被基因解剖和 必要的监管成分将被识别、分离、纯化和 特色化的。建议的工作包括分离缺失突变体 克隆并对这些克隆进行互补测试以定义数量 所涉及的监管组成部分；监管部门的标识 利用基因融合技术(插入lac Z基因)开发的蛋白质 调控基因核苷酸序列的测定； 构建高效生产调节蛋白的克隆，以便它们 可方便地大量分离用于生化 工作；DNA结合中调节蛋白的生化特征， 抑制子和激活子的相互作用及诱导剂对DNA的影响 结合以及阻遏子和激活子之间的相互作用 体外转录和转录-翻译耦合系统。这个 这个项目的最终目标是充分了解分子和 外源诱导剂诱导基因表达的生化机制。

The expression of the phosphoglycerate transport (pgt) system of Salmonella typhimurium is induced by exogenous, but not endogenous, inducers. We have cloned the pgt system into Escherichia coli using the plasmid pBR322. The cloned DNA contains all regulatory elements needed since the cloned system is regulated identically as in S. typhimurium. Preliminary work from this laboratory indicates that the operon is negatively controlled and there exists repressor and activator which counteracts and neutralizes the action of repressor. Because of the novelty of exogenous induction of gene expression, the regulatory machinery will be genetically dissected and necessary regulatory components will be identified, isolated, purified and characterized. The proposed work involves isolation of deletion mutant clones and performing complementation test on these clones to define number of regulatory components involved; identification of the regulatory proteins using gene fusion technique (insertion of lac Z gene) developed by Beckwith; determination of nucleotide sequence of the regulatory genes; construction of clones hyperproducing the regulatory proteins so that they can be conveniently isolated in large quantity to be used for biochemical work; biochemical characterization of regulatory proteins in DNA binding, interaction between repressor and activator, effect of inducers on the DNA binding and the repressor and activator interaction to be carried out in in vitro transcription and coupled transcription-translation systems. The ultimate goal of this project is to fully understand the molecular and biochemical mechanism of the gene expression induced by exogenous inducers.