MECHANISMS OF RADIATION INDUCED GENOMIC INSTABILITY

辐射引起的基因组不稳定的机制

• 批准号: 2377297
• 负责人: ANDREW J. GROSOVSKY
• 金额: $ 19.87万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-10 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2377297
• 关键词: CHO cells; DNA damage; aminopurine; artificial chromosomes; centromere; chromatin; chromosome aberrations; cytogenetics; fluorescent in situ hybridization; gene rearrangement; genome; heterochromatin; karyotype; nucleic acid sequence; phenotype; protein kinase; radiation genetics; transfection

This proposal is designed to investigate mechanism of radiation induced genetic instability (GI) in human cells. The overall hypothesis is that GI is associated with a critical structural alteration of a chromosome which is sensitive to further breakage and rearrangement, rather than a mutation of a specific gene. Furthermore, we postulate that chromosomal rearrangements involving the novel juxtaposition of euchromatin with centromeric (alpha) or pericentromeric (classical) heterochromatin is one such critical structural alteration. Sub-clonal analysis of persistent radiation induced instability will be utilized to test the hypothesis that elevated rates of delayed chromosomal rearrangements, mutation at the tk locus, and delayed reproductive cell death, varies among sub-clones from the same parent. We will specifically determine if sub-clones including complex chromosomal abnormalities will exhibit higher rates of persistent GI than other sub-clones which do not include a complex abnormality due to karyolytic heterogeneity in the parental clone. 2,6- diaminopurine (DAP) will be utilized as a reagent which induces highly preferential breakage in the alpha and classical heterochromatin of chromosomes 1, 9 and 16. The high specificity and frequency of DAP induced damage, and the availability of easily visualized fluorescent probes for the affected heterochromatic sequences, will enable a prospective analysis of the emergence of GI by focused examination of the fate of damaged heterochromatin. This system will also be utilized to test the hypothesis that persistent chromosomal instability will be influenced by cellular capacity for double strand break (dsb) repair. An APRT derivative of the DNA-dependent protein kinase catalytic sub unit (DNA-PK) Cho cell mutant, V-3, will be isolated and restored to radio resistance by instruction of the XRCC7 gene which encode DNA-PK. This cell, and its isogeneic non-transfected counterpart, will be used as recipients for the transfer of abnormal or control clonal copies of chromosome 16 from DAP treated TK6 cells, using a prt (16q24.3) as a selectable marker. The rate of instability of these chromosomes will be compared in the two recipient cell lines, and to the ongoing rate of instability of the same chromosomes in their native cellular environment.

该建议旨在研究辐射诱导的机制 人类细胞中的遗传不稳定性（GI）。 总体假设是GI 与染色体的关键结构改变有关 对进一步的破裂和重排敏感，而不是突变 特定基因。 此外，我们假设该染色体 重排涉及新颖的斑塑素并置与 centromeric（Alpha）或丁香粒（经典）异染色质是一个 这种关键的结构改变。 持久性的亚克隆分析 辐射引起的不稳定性将用于检验以下假设。 延迟染色体重排的速度升高，突变在 TK基因座和延迟的生殖细胞死亡在亚clones中有所不同 来自同一父母。 我们将明确确定是否子卡隆 包括复杂的染色体异常将表现出更高的速率 持续的GI比其他不包括复杂的子clones 父母克隆中的核溶液异质性引起的异常。 2,6- 二氨嘌呤（DAP）将被用作高度诱导的试剂 α和经典异染色质的优先断裂 染色体1、9和16。DAP的高特异性和频率 诱导的损坏以及易于可视化荧光的可用性 受影响的异质序列的探针将启用a 通过重点检查GI出现的前瞻性分析 受损异染色质的命运。 该系统也将被使用 测试持续性染色体不稳定性的假设将是 受双链断裂（DSB）修复的细胞容量的影响。 一个 DNA依赖性蛋白激酶催化子单位的APRT衍生物 （DNA-PK）CHO细胞突变体V-3将分离并恢复到无线电 通过编码DNA-PK的XRCC7基因的指示抗性。 该单元及其异质性非转染对应物将被用作 转移异常或对照克隆副本的受体 DAP处理过的TK6细胞的16染色体，使用PRT（16q24.3）作为 可选标记。 这些染色体的不稳定率将 在两个接受者细胞系中进行比较，并持续 同一染色体在其天然细胞环境中的不稳定性。