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EVOLUTION AND REGULATION OF MERCURIAL RESISTANCE GENES

耐汞基因的进化和调控

基本信息

批准号：
3466028
负责人：
TAPAN K MISRA
金额：
$ 11.61万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-16 至 1994-08-31
项目状态：
已结题

项目摘要

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.

汞离子和汞的转录调控和基因调控革兰氏阳性质粒的牛至汞抗性操纵子将对革兰氏阴性菌进行研究。调控基因从广谱的汞电阻操纵子粘质沙雷氏菌pDU1358与金黄色葡萄球菌将pI258克隆到高效表达载体(PI258)中在大肠杆菌中。过度表达的调节蛋白将是纯化及其与汞抗性操纵子的相互作用将通过凝胶结合、硝酸纤维过滤器结合和羟基自由基足迹技术。的开始站点调控和结构基因的转录将是下定决心。调节蛋白对体外培养的大鼠心脏功能的影响转录后的信使核糖核酸结构基因将被转录在有机汞化合物存在和不存在的情况下进行研究径流转录分析。预测的螺旋的相互作用- 调控蛋白与操纵子的转角螺旋结构 DNA将进行检测，并对这种结构提出绝对要求与DNA的特异性结合将通过位点定向来确认诱变研究。特定的核苷酸序列，使与调节蛋白的DNA结合域接触将要下定决心。参与汞离子转运的基因和金黄色葡萄球菌质粒pI258中的有机汞将被以使用寡核苷酸进行分子遗传学研究为特征的定位突变和亚硫酸氢钠诱导的定位突变。