MUTAGENESIS

诱变

• 批准号: 6993340
• 负责人: HOWARD L LIBER
• 金额: $ 21.92万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6993340
• 关键词: DNA damage; cell components; cell line; cell nucleus; congenital aplastic anemia; cytotoxicity; flow cytometry; free radical oxygen; gene mutation; gene targeting; mutagens; oxidative stress; p53 gene /protein; pharmacokinetics; photoactivation; photosensitizing agents

The goal of this project is to further understanding of how short-lived reactive oxidizing species (ROS) generated outside the nucleus nevertheless lead to the induction of gene locus mutations. To accomplish this, this project will investigate quantitative and qualitative aspects of the induction of gene mutations by ROS generated outside the nucleus, and compare them to mutations generated by ROS within the nucleus. ROS will be produced uniformly within the cell or targeted to non-nuclear regions by using photo-activated dyes that concentrate in specific sub-cellular compartments. Quantitative and qualitative measurements of DNA damage will be made using the comet, or single ceil gel electrophoresis assay. The mutagenicity studies will involve a two-pronged approach. First, quantitative dose-response experiments will be carried out at two endogenous gene loci, and measured as a function of damage to DNA. Second, the qualitative natures of induced mutants will be determined by analyses of mutational spectra induced by the various treatments. These studies will be done initially in human lymphoblastoid cells with a mutated p53 gene. An additional set of experiments will address the question of whether mutagenic effects of ROS exposure are dependent on p53 status. The same strategies described above will be repeated using p53 wild type and null cells from the same donor. Finally, another set of experiments will examine effects of the Fanconi's anemia complex on ROS mutagenesis.

该项目的目标是进一步了解短命活性氧化物质如何 然而，在细胞核外产生的（ROS）会导致基因位点的诱导 突变。为了实现这一目标，该项目将调查定量和定性方面 细胞核外产生的 ROS 诱导基因突变，并将它们与 细胞核内ROS产生的突变。 ROS会在细胞内均匀产生 或通过使用集中在特定区域的光激活染料来靶向非核区域 亚细胞区室。 DNA 损伤的定量和定性测量将 使用彗星或单细胞凝胶电泳测定法进行。致突变性研究将 涉及双管齐下的方法。首先，将进行定量剂量反应实验 两个内源基因位点，并作为 DNA 损伤的函数进行测量。其次， 诱导突变体的定性将通过突变谱分析来确定 由各种治疗引起。这些研究最初将在人类淋巴母细胞中进行 具有突变 p53 基因的细胞。一组额外的实验将解决以下问题 ROS 暴露的致突变作用是否取决于 p53 状态。将使用来自同一供体的 p53 野生型和无效细胞重复上述相同的策略。 最后，另一组实验将检查范可尼贫血综合症对 ROS诱变。