EVOLUTION AND REGULATION OF MERCURIAL RESISTANCE GENES

汞离子对转录和基因调控的调控 革兰氏阳性菌质粒的牛至汞抗性操纵子 和革兰氏阴性菌进行研究。 调控基因 从广谱抗汞操纵子的 粘质沙雷氏菌质粒pDU 1358和金黄色葡萄球菌 将质粒pI 258克隆到超表达载体（pI 258）中， 在大肠杆菌中。 过度表达的调节蛋白将被 纯化的及其与抗汞操纵子的相互作用 将通过凝胶结合、硝化纤维素过滤结合和 羟基自由基足迹技术。 开始网站的 调控和结构基因的转录将被 测定 调节蛋白对体外细胞凋亡的影响 转录的mRNA转录的结构基因将是 在存在和不存在有机汞的情况下， 径流转录测定。预测的螺旋的相互作用- 具有操纵子的调节蛋白的转角-螺旋结构 DNA将被测试和绝对要求这样的结构， 与DNA的特异性结合将通过位点定向的 诱变研究。 特定的核苷酸序列， 与调节蛋白的DNA结合结构域的接触将 被确定。 参与汞离子转运的基因， 金黄色葡萄球菌质粒pI 258中的有机汞 通过使用寡核苷酸的分子遗传学研究来表征 定点突变和亚硫酸氢钠诱导局部突变。

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.