OXYGEN AND SPONTANEOUS MUTAGENESIS IN ESCHERICHIA COLI

描述：(改编自调查人员摘要)活性物种 氧气通常是由呼吸细胞产生的，众所周知是有毒的， 破坏蛋白质、膜脂和DNA。这些有毒的副产品 有氧呼吸，包括超氧阴离子自由基、过氧化氢 和羟基自由基，被认为可能是 诱变、致癌和衰老。虽然详细的路径是通过 造成上述条件的氧物种尚未明确定义， 与DNA的相互作用是一个明显的组成部分。在多种形式的 DNA损伤，突变事件的产生是由基因决定的 分析。 使用基因特征良好的兼性好氧细菌， 大肠埃希氏菌正向(LacI-、利福平和 Valine抗性)和Back(TRPA+)突变频率揭示了 可归因于氧气的自发突变的比例。 好氧和厌氧(介质减少的厌氧室)条件将 揭示了自发突变的比例可以归因于 氧气。需氧和厌氧突变谱将使用以下方法确定 TRPA逆转系统和LacI(-)突变的DNA测序 光谱提供了对突变机制的洞察。好氧和厌氧 将测量突变E。 具有正常保护作用的酶活性缺陷的大肠杆菌菌株 氧化损伤(过氧化氢酶{katG，kat}和超氧化物歧化酶{苏打， SodB)和DNA修复(recA、polA、xthA和muL)。厌氧诱变 频率和频谱将使用最稳定的 活性氧物种，过氧化氢，作为诱变剂。 这个项目的目标是：(1)比较好氧和无氧 大肠杆菌中的突变频率以确定氧的贡献 物种朝向自发突变的负载，(2)定义突变 野生型和突变型大肠杆菌菌株的光谱，以提供 氧诱导突变机制，以及(3)阐明机制(S) 过氧化氢诱导的诱变作用。

DESCRIPTION: (adapted from the investigators abstract) Active species of oxygen are normally generated by respiring cells and are known to be toxic, damaging proteins, membrane lipids and DNA. These toxic byproducts of aerobic respiration, which include superoxide radical, hydrogen peroxide and hydroxyl radical, are thought to be possible causative agents in mutagenesis, carcinogenesis and aging. Although detailed pathways by which oxygen species cause the above conditions have yet to be clearly defined, interaction with DNA is an obvious component. Among the multiple forms of DNA injury, the production of mutational events is amenable to genetic analysis. Using the genetically well-characterized facultative aerobic bacterium, Escherichia coli, measurement of forward (LacI-, rifampin resistance and valine resistance) and back (TrpA+) mutation frequencies reveal the proportion of spontaneous mutagenesis that can be attributed to oxygen. Aerobic and anaerobic (anerobic chamber with reduced media) conditions will reveal the proportion of spontaneous mutagenesis that can be attributed to oxygen. Aerobic and anaerobic mutational spectra will be determined using the trpA reversion system and the DNA sequencing of lacI(-) mutations; such spectra provide insights into mutational mechanisms. Aerobic and anaerobic frequencies (and possibly mutational spectra) will be measured in mutant E. coli strains defective in enzymatic activity that normally protects against oxidative damage (catalase {katG, kat} and superoxide dismutase {sodA, sodB}) and DNA repair (recA, polA, xthA and mutL). Anaerobic mutation frequencies and spectra will be determined using the most stable of the active oxygen species, hydrogen peroxide, as a mutagen. The goals of this project are to: (1) compare aerobic and anaerobic mutation frequencies in E. coli to establish the contribution of oxygen species toward the load of spontaneous mutations, (2) define mutational spectra in wild type and mutant E. coli strains, to provide models of oxygen-induced mutational mechanisms, and (3) elucidate the mechanisms(s) of hydrogen peroxide-induced mutagensis.