IONIZING RADIATION--INDUCED RECOMBINATION

电离辐射——诱导复合

• 批准号: 2384926
• 负责人: Jac A Nickoloff
• 金额: $ 17.87万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2384926
• 关键词: DNA repair; gene targeting; genetic mapping; genetic recombination; human tissue; ionizing radiation; linear energy transfer; mouse mammary tumor virus; mutant; neomycin; radiation dosage; radiation sensitivity; radiobiology; restriction fragment length polymorphism

The goals of the proposed research are to explore the effects of ionizing radiation-induced interchromosomal recombination in normal human cells and in mutant cells with altered radiosensitivity and recombination phenotypes. Few studies have focused on the recombinogenic effects of ionizing radiation in mammalian cells, and thus far none have examined such effects as a function of dose rate, linear energy transfer (LET), or cellular repair capacity. It has been difficult to study the mechanisms of ionizing radiation-induced recombination in mammalian cells because of difficulties in analyzing recombinant products. Furthermore, although ionizing radiation is known to induce homologous recombination between alleles present at homologous autosomal loci in mammalian cells, it is not known whether this is due to direct effects of damage in recombining genes (i.e., strand breaks) or indirect effects (i.e., by inducing enzymes involved in recombinational repair) or both. In the yeast Saccharomyces cerevisiae, ionizing radiation-induced recombination is thought to involve both direct and indirect processes. This proposal describes model systems that will provide information about the induction of recombination in human cells by radiations of various types. The proposed recombination substrates will consist of noncomplementing (heteroallelic) neomycin (neo) genes targeted to both copies of an autosomal locus, and will include several features. Recombination substrates will be flanked by convenient restriction sites and neo alleles will be heteroallelic at eleven positions consisting of phenotypically silent restriction fragment length polymorphisms (RFLPs). The flanking restriction sites and RFLPs will facilitate the rescue and fine-resolution mapping of recombinant products. The neo alleles will be regulated by the inducible mouse mammary tumor virus (MMTV) promoter, allowing experimental control of transcription rates in recombination substrates. Complementary approaches are proposed to address questions about how recombination induces recombination. One approach involves the detailed examination of spontaneous and radiation- induced recombination products of defined heteroallelic genes, including studies of the influence of transcription on induced recombination frequencies and mechanisms. Such analyses will provide increased sensitivity for detecting potential mechanistic differences among spontaneous, low LET-induced and high LET-induced recombination events. In another approach we will investigate whether induced recombination is a consequence of direct effects of damage in recombining regions and/or an indirect induction of recombinational repair enzymes. Lastly, comparisons will be made between spontaneous and radiation-induced recombination frequencies and product spectra in DNA repair-proficient normal human cells, and DNA repair-deficient cells from patients suffering from ataxia- telangiectasia and Bloom's syndrome. These experiments will help to establish the roles of the gene products altered in these mutant cells. Together these studies will further our understanding of the initiation, mechanisms, and genetic consequences of spontaneous and ionizing radiation-induced recombination in mammalian cells.

拟议研究的目标是探索电离的影响 正常人体细胞中辐射诱导的染色体间重组 在放射敏感性和重组发生改变的突变细胞中 表型。很少有研究关注重组效应 哺乳动物细胞中的电离辐射，迄今为止还没有人检查过 这种效应是剂量率、线性能量转移 (LET) 或 细胞修复能力。研究其机制一直很困难 电离辐射诱导哺乳动物细胞重组的原因 分析重组产物的困难。此外，虽然 已知电离辐射会诱导之间的同源重组 等位基因存在于哺乳动物细胞的同源常染色体位点，但不是 已知这是否是由于重组基因损伤的直接影响 （即链断裂）或间接影响（即通过诱导酶 参与重组修复）或两者兼而有之。 在酵母菌中 在酿酒酵母中，电离辐射诱导的重组被认为涉及 直接和间接过程。该提案描述了模型系统 这将提供有关重组诱导的信息 人体细胞受到各种类型的辐射。拟议的重组 底物将由非互补（异等位基因）新霉素 (neo) 组成 针对常染色体基因座的两个副本的基因，并且将包括 几个特点。 重组底物两侧将有方便的 限制性位点和新等位基因在 11 处将是异等位基因 由表型沉默限制性片段长度组成的位置 多态性（RFLP）。侧翼限制位点和 RFLP 将 促进重组产物的拯救和精细分辨率图谱。 neo等位基因将受到诱导性小鼠乳腺肿瘤的调节 病毒（MMTV）启动子，允许实验控制转录 重组底物的速率。提出了补充方法 解决有关重组如何引发重组的问题。 一 方法包括对自发和辐射的详细检查 确定的异等位基因的诱导重组产物，包括 转录对诱导重组影响的研究 频率和机制。 此类分析将提供更多 检测潜在机制差异的灵敏度 自发的、低 LET 诱导的和高 LET 诱导的重组事件。 在另一种方法中，我们将研究诱导重组是否是 重组区域和/或损伤的直接影响的结果 重组修复酶的间接诱导。 最后，比较 将在自发重组和辐射诱导重组之间进行 DNA 修复能力强的正常人的频率和产物光谱 细胞和来自共济失调患者的 DNA 修复缺陷细胞 毛细血管扩张和布卢姆综合征。这些实验将有助于 确定这些突变细胞中改变的基因产物的作用。 这些研究将进一步加深我们对启动的理解， 自发和电离的机制和遗传后果 哺乳动物细胞中辐射诱导的重组。