EVOLUTION AND REGULATION OF MERCURIAL RESISTANCE GENES

耐汞基因的进化和调控

• 批准号: 3466027
• 负责人: TAPAN K MISRA
• 金额: $ 11.19万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-16 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466027
• 关键词: DNA binding protein; DNA footprinting; Serratia marcescens; Staphylococcus aureus; bacterial genetics; biochemical evolution; gene induction /repression; genetic mapping; genetic transcription; gram negative bacteria; gram positive bacteria; ion transport; mercury; messenger RNA; molecular cloning; mutant; nucleic acid sequence; operon; regulatory gene; structural genes

Control of transcription and gene regulation in mercuric ion and oregano-mercurial resistance operons of plasmids from Gram positive and Gram negative bacteria will be studied. The regulatory genes from the broad spectrum mercurial resistance operons of the Serratia marcescens plasmid pDU1358 and Staphylococcus aureus plasmid pI258 will be cloned into a hyperexpression vector (pI258) in Escherichia coli. Overexpressed regulatory proteins will be purified and their interactions with mercurial resistance operons will be studied by gel binding, nitrocellulose filter binding and hydroxyl radical footprinting techniques. Start sites of the transcriptions of the regulatory and structural genes will be determined. The effects of the regulatory protein on the in vitro transcription of mRNA transcribed the structural genes will be studied in the presence and absence of and organomercurials using run-off transcription assays. Interaction of the predicted helix- turn-helix structure of the regulatory proteins with the operator DNA will be tested and absolute requirement of such a structure for specific binding with the DNA will be confirmed by site-directed mutagenesis studies. Specific nucleotide sequences that make contact with the DNA binding domain of the regulatory proteins will be determined. Genes involved in the transport of mercuric ion and organomercurials in Staphylococcus aureus plasmid pI258 will be characterized by molecular genetic studies using oligonucleotide site directed and sodium bisulfite induced localized mutations.

汞离子和基因调控的转录和基因调控 革兰氏阳性质粒的牛至汞抗性操纵子 将研究革兰氏阴性细菌。 调控基因 来自广谱汞抗性操纵子 粘质沙雷氏菌质粒 pDU1358 和金黄色葡萄球菌 质粒 pI258 将被克隆到超表达载体 (pI258) 在大肠杆菌中。 过度表达的调节蛋白将 纯化及其与耐汞操纵子的相互作用 将通过凝胶结合、硝化纤维滤膜结合和 羟基自由基足迹技术。 的启动站点 调节基因和结构基因的转录将是 决定。 调节蛋白对体外的影响 mRNA的转录 转录的结构基因将是 在存在和不存在有机汞的情况下进行了研究 径流转录测定。预测螺旋的相互作用- 调节蛋白与操纵子的螺旋结构 DNA将被测试并且绝对需要这样的结构 与 DNA 的特异性结合将通过定点确认 诱变研究。 特定的核苷酸序列使得 与调节蛋白的 DNA 结合域接触将 被确定。 参与汞离子运输的基因 金黄色葡萄球菌质粒 pI258 中的有机汞将 使用寡核苷酸进行分子遗传学研究 定点突变和亚硫酸氢钠诱导的局部突变。