EFFECTS OF SELECTED METAL SALTS ON THE FIDELITY OF DNA SYNTHESIS IN VITRO

暴露于微量金属的生理效应 在工作场所和环境中的行为都有详细记录。 几种金属 被认为是诱变剂和致癌物。 值得注意的是，Mn，Co，Cd， 已发现Ag、Ba、Cr、Pb、Ni和As增加碱取代 在体外DNA复制试验中至少有30%的误差， 检测错误配对的核苷酸相对于 合成多核苷酸DNA模板。 本研究拟全面分析其致突变作用， 金属盐对DNA合成保真度的影响将采用 体外保真度测定。该测定允许检测多种 在250个核苷酸序列的合成过程中出现的不同错误 包含在天然DNA模板中。 利用这个系统，我们将 系统地研究了各种金属盐对 体外DNA合成的保真度。 具体来说，我们将测量剂量 所选金属对体外DNA合成和保真度的影响， 确定特定类型的突变频率和错误率 突变，并构建所选金属的突变谱。 的 这项工作的结果预计将提供有关以下方面的宝贵信息 所选金属的诱变特异性和剂量水平，并且可以 提供了对金属诱导的诱变机制的进一步了解。 这个项目将包括学生在各个方面。学生将学习 DNA操作基础，空斑分析，体外DNA 复制、微生物学技术、DNA测序、数据管理， 以及危险金属盐和放射性核素的安全使用。

The physiological effects of exposure to trace quantities of metals in the workplace and in the environment are well documented. Several metals have been identified as mutagens as well as carcinogens. Notably, Mn, Co, Cd, Ag, Ba, Cr, Pb, Ni, and As have been found to increase base substitution errors by at least 30% in an in vitro DNA replication assay designed to detect misincorporation of an incorrectly paired nucleotide opposite a synthetic polynucleotide DNA template. In this proposal, a comprehensive analysis of the mutagenetic effects of metal salts on the fidelity of DNA synthesis will be undertaken using an in vitro fidelity assay. The assay allows detection of a variety of different errors occurring during synthesis of a 250 nucleotide sequence contained within a natural DNA template. Utilizing this system, we will systematically study the effects of a variety of metal salts on the fidelity of in vitro DNA synthesis. Specifically, we will measure dose effects of the selected metals on in vitro DNA synthesis and fidelity, determine mutation frequencies and error rates for specific types of mutations, and construct mutational spectra for the selected metals. The results of this work are expected to provide valuable information on mutagenic specificity and dose levels for the selected metals, and may provide further insight into the mechanism of metal-induced mutagenesis. This project will include students in every aspect. Students will learn fundamentals of DNA manipulations, plaque assays, in vitro DNA replication, microbiological techniques, DNA sequencing, data management, and safe use of hazardous metal salts and radionuclides.