Mechanisms of Radiation-Induced Genomic Istability

描述(由申请人提供)： 这份修订后的申请是对一项调查提案的竞争性更新 辐射引起的基因组不稳定性。此应用程序的目标是 巩固“不稳定性计划”，重点研究分子机制和 辐射引起的不稳定性的生物学后果。建议进行的研究 是PI实验室当前研究的扩展，并将建立 根据上一次供资期间的观察结果。我们建议 继续验证基于质粒的重组试验作为一种快速和 辐射后细胞延迟不稳定的可靠指标。 此外，我们将利用广泛收集的染色体不稳定 我们在前一个资助期内产生的细胞克隆 研究不稳定性在辐射抗性中的作用并确定 基因表达的差异可能是导致观察到的 不稳定。要实现这些目标，有三个具体目标： 特定目标1将测试基于以下条件的重组测试的假设 绿色荧光蛋白的表达可以快速、高效地用于 识别不稳定克隆作为辐射后时间的函数。我们会 描述这个基于质粒的检测系统与我们的油井的关系 定义染色体分析，并用它来确定分离的效果 放射治疗、DNA修复抑制剂和照射后修饰 关于诱发不稳定性。 特定目标2将在我们观察到基因组不稳定性的基础上进行扩展 对细胞辐射抗性的贡献，我们将研究这一假说 这是由于基因表达的差异造成的。 特定目标3将测试辐射诱导基因组的假设 不稳定具有反映差异基因的分子/遗传基础 以及差异显示策略和/或RNA杂交 利用微阵列技术的策略可以用来鉴定基因(S) 参与启动和/或保持不稳定的表型。

DESCRIPTION (provided by applicant): This revised application is a competing renewal of a proposal to investigate radiation-induced genomic instability. The goal of this application is to consolidate the "instability program" and focus on the molecular mechanisms and biological consequences of radiation-induced instability. The proposed studies are an expansion of current research from the PI's laboratory and will build upon observations made during the previous funding period. We propose to continue to validate a plasmid based recombination assay as a rapid and reliable indicator of delayed instability in cells post irradiation. Furthermore, we will utilize the extensive collection of chromosomally unstable clones of cells we have generated during the previous funding period to investigate the role of instability in radioresistance and determine differences in gene expression that may be responsible for the observed instability. To accomplish these goals there are three specific aims: Specific aim 1 will test the hypothesis that a recombination assay based on expression of green fluorescence protein can be used to rapidly and efficiently identify unstable clones as a function of time after irradiation. We will characterize this plasmid based assay system in relationship to our well defined chromosomal assay and use it to determine the effects of fractionated radiation treatment, DNA repair inhibitors, and modifying the post irradiation on induced instability. Specific aim 2 will expand upon our observation that genomic instability contributes to cellular radioresistance and we will investigate the hypothesis that this is due to differential gene expression. Specific aim 3 will test the hypothesis that radiation-induced genomic instability has a molecular/genetic basis reflecting differential gene expression, and that differential display strategies and/or RNA hybridization strategies using microarray technology can be used to identify the gene(s) involved in initiating and/or perpetuating the unstable phenotype.