RADIATION INDUCED GENETIC ALTERATIONS IN MAMMALIAN CELLS

由电离辐射引发的分子事件导致基因 将对哺乳动物细胞的损伤和致癌进行研究。我们计划 构建重组DNA分子，将部分 牛乳头状瘤病毒基因组(它将提供一个复制子和可选择的 能够在哺乳动物细胞中发挥作用的标记)，大肠杆菌的一部分 质粒pBR322(它将提供复制子和可选择的标记 在大肠杆菌中发挥作用)和大肠杆菌lac I基因(这将作为 特定遗传事件发生的指示器)。结构性 将在lac i基因中引入包括point在内的修饰 突变、短串联复制、移码突变和插入 外来DNA。含有突变I基因的质粒将被引入到 小鼠C127I细胞；转化的细胞灶将被分离并用于 放射治疗对回归野外的影响分析 I型基因表型。突变I基因的逆转将通过以下方式进行评分 转化适当的指示菌株和后续 含显色底物平板上菌落的评价。 或者，细菌裂解物将被检测为lac抑制物。 蛋白。电离辐射对不同类型逆转率的影响 将调查损伤的影响(包括剂量、剂量率和 辐射质量)。可诱导的突变或重组功能将 通过将辐照细胞与未处理的细胞融合进行评估 含有突变I基因的质粒组。突变人类的能力 成纤维细胞(例如来自共济失调毛细血管扩张症患者的细胞)对 DNA损伤将通过类似的融合实验进行评估。含有DNA的 将检查辐射引起的损伤或其他特定的修改 通过将这种DNA导入携带突变质粒的细胞并检测 用于诱变或重组的状态。最后， 受辐射细胞提取物引起特定基因的能力 更改情况将会确定。这项研究的长期目标是 了解细胞如何对DNA损伤做出反应，以及这种反应起到什么作用 在致癌过程中起作用。我们提出的化验方法有希望 提供有关完整的分子事件的特定信息 细胞。

Molecular events initiated by ionizing radiation which result in genetic damage and carcinogenesis in mammalian cells will be investigated. We plan to construct recombinant DNA molecules which combine a portion of the bovine papilloma virus genome (which will provide a replicon and selectable marker capable of functioning in mammalian cells), a portion of E. coli plasmid pBR322 (which will provide a replicon and selectable marker capable of functioning in E. coli) and the E. coli lac i gene (which will serve as an indicator for the occurrence of specific genetic events). Structural modifications will be introduced into the lac i gene including point mutations, short tandem duplications, frameshift mutations and insertions of foreign DNA. Plasmids containing mutant i genes will be introduced into mouse C127I cells; transformed cell foci will be isolated and used for analysis of the effects of radiation-treatment on reversion to the wild type i gene phenotype. Reversion of mutant i genes will be scored by transforming the appropriate indicator bacterial strain and subsequent evaluation of colonies on plates containing chromogenic substrate. Alternatively, bacterial lysates will be assayed for lac repressor protein. Effects of ionizing radiation on reversion of the various types of lesions will be investigated (including effects of dose, dose rate and radiation quality). Inducible mutagenic or recombinogenic functions will be evaluated by fusing irradiated cells with untreated cells carrying plasmids containing mutant i genes. The ability of mutant human fibroblasts (e.g. cells from ataxia telangiectasia patients) to respond to DNA damage will be evaluated by similar fusion experiments. DNA containing radiation-induced lesions or other specific modifications will be examined by introducing this DNA into cells carrying the mutant plasmids and testing for the induction of a mutagenic or recombinogenic state. Finally, the ability of extracts from irradiated cells to cause specific genetic alterations will be determined. The long range goal of this research is to understand how cells respond to DNA damage and what role this response plays in carcinogenesis. The assay we propose holds the promise of providing specific information on molecular events which occur in intact cells.