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 辐射诱导的正常人细胞和 在变化的放射敏感和重组的突变细胞中 表型。很少有研究集中于重组作用 哺乳动物细胞中的电离辐射，到目前为止都没有检查 此类效果，包括剂量率，线性能量转移（LET）或 细胞修复能力。很难研究 由于哺乳动物细胞的电离辐射诱导的重组 分析重组产品的困难。此外，虽然 已知电离辐射会诱导同源重组 哺乳动物细胞中同源常染色体基因座的等位基因，不是 知道这是否是由于重组基因的直接影响 （即链断裂）或间接效应（即，通过诱导酶 参与重组维修）或两者兼而有之。 在酵母菌中 酿酒酵母，电离辐射诱导的重组被认为涉及 直接和间接过程。该建议描述了模型系统 这将提供有关诱导重组的信息 人类细胞通过各种类型的辐射。提出的重组 底物将包括不合格（异源）新霉素（NEO） 针对常染色体基因座的两个副本的基因，并将包括 几个功能。 重组底物将侧翼是方便的 限制位点和NEO等位基因将在11处是异性词 由表型上沉默限制片段长度组成的位置 多态性（RFLP）。侧翼限制站点和RFLP将会 促进重组产品的救援和精细分辨率映射。 NEO等位基因将受诱导小鼠乳腺肿瘤调节 病毒（MMTV）启动子，允许对转录的实验控制 重组底物的速率。提出了补充方法 解决有关重组如何诱导重组的问题。 一 方法涉及对自发和辐射的详细检查 - 诱导的定义杂同基因的重组产物，包括 研究转录对诱导重组的影响 频率和机制。 这样的分析将增加 检测潜在机械差异的敏感性 自发的，低的诱导且高诱导的重组事件。 在另一种方法中，我们将研究诱导的重组是否是 重新组合区域和/或一个直接损害影响的结果 间接诱导重组修复酶。 最后，比较 将在自发和辐射引起的重组之间进行 DNA修复良好的正常人中的频率和产品光谱 细胞和DNA修复缺陷的细胞，来自共济失调的患者 Telangiectia和Bloom综合征。这些实验将有助于 建立在这些突变细胞中改变的基因产物的作用。 这些研究共同将进一步了解我们对启动的理解， 机理和自发和电离的遗传后果 辐射诱导的哺乳动物细胞中的重组。