SPECIFICITY OF OXYGEN DNA DAMAGE AND MUTAGENESIS

氧 DNA 损伤和诱变的特异性

• 批准号: 6097923
• 负责人: LAWRENCE A LOEB
• 金额: $ 15.89万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6097923
• 关键词: Aves; DNA damage; DNA repair; Werner's syndrome; aging; clinical research; congenital aplastic anemia; flow cytometry; free radical oxygen; gene mutation; genetically modified animals; human subject; laboratory mouse; lymphocyte; mutagens; oxidative stress; polymerase chain reaction; species difference

Our goal for the proposed grant period is to ascertain whether oxygen free radical-induced damage accumulates in DNA during aging, and whether capacity to repair this damage diminishes with age. During the previous grant period, we identified lesions in DNA, i.e., characteristic chemical modifications and mutations, that provide specific markers for genomic alterations caused by oxygen free radicals. We have three specific aims. Having established that CC->TT tandem substitutions are produced in bacteria exposed to oxygen free radicals, we will utilize a new PCR-based assay to quantitate the frequency of CC->TT mutations in mammalian cells and tissues. Having documented that 5-hydroxy 2'-deoxycytidine is produced by oxygen free radicals and is highly mutagenic in bacteria, we will measure the content, repair and mutagenicity of this adduct in mammalian cells. Having introduced a flow cytometric assay to measure DNA damage during the cell cycle, we will utilize this assay to quantitate oxygen- mediated DNA damage and repair in cells and tissues. We will obtain tissue from mice (ages 6-8, 16-18 and over 24 months), as well as kidney and lymphocytes from human subjects of various ages. We will also utilize lymphoblast cell lines from patients with Fanconi's anemia which are uniquely sensitive to oxygen damage. These model systems are established in the laboratories that constitute this program project grant. We will also obtain tissue from transgenic mice that overexpress human catalase alone or both human SOD-1 and catalase, and from avian tissue as it becomes available from project 1. We will utilize the three assays mentioned in the specific aims to examine these model systems. Our observations will allow us to assess the accumulation of oxidative damage to DNA as a function of age and oxidative state in three species.

我们提议的赠款期的目标是确定是否无氧 自由基诱导的损伤在衰老过程中会积聚在DNA中，以及是否是否 维修这种损害的能力随着年龄的增长而减少。在上一个 赠款期，我们确定了DNA中的病变，即特征化学 修改和突变，为基因组提供特定标记 由氧自由基引起的改变。我们有三个具体的目标。 确定CC-> tt串联替换是在 暴露于氧气自由基的细菌，我们将利用一种新的基于PCR的 测定量化哺乳动物细胞中CC-> TT突变的频率 和组织。记录了5-羟基2'-脱氧胞丁丁的 通过氧气自由基，在细菌中是高度诱变的，我们将 测量此加合物在哺乳动物中的含量，修复和诱变性 细胞。引入了流式细胞仪测定以测量DNA损伤 在细胞周期中，我们将利用该测定法来定量氧气 细胞和组织中介导的DNA损伤和修复。 我们将获得 小鼠的组织（6-8岁，16-18岁和24个月），以及肾脏 以及来自各个年龄的人类受试者的淋巴细胞。我们还将利用 来自法科尼贫血患者的淋巴细胞细胞系 对氧损伤的独特敏感。这些模型系统是建立的 在构成该计划项目赠款的实验室中。我们将 还从过表达人过氧化氢酶的转基因小鼠中获得组织 单独或人类SOD-1和过氧化氢酶，以及从鸟类组织中 从项目1获得。我们将利用这三个测定法 在检查这些模型系统的具体目的中提到的。我们的 观察结果将使我们能够评估氧化损伤的积累 在三种物种中，DNA作为年龄和氧化状态的函数。