X-RAY DAMAGE & REPAIR OF PRIMATE CELL & DNA SEQUENCES

检查X射线对细胞DNA的损伤和修复 序列水平需要获得DNA的同质群体 辐射细胞的序列。 我们证明了 这在研究高度重复的α组分中的可行性 从经辐照的猴CV-1细胞制备的DNA序列。 的 系统用于评估α修复的时间过程 DNA和咖啡因对修复的影响。 我们现在将研究范围扩大到辐射后的 质粒转染到宿主细胞中。 这种方法允许 均匀布居的x射线损伤与修复研究 克隆基因 最终，它应该提供宿主细胞反应- 用于检测和评估 单个DNA修复酶和转染的基因， 为他们 质粒作为细胞DNA的替代靶点。 在转染前验证质粒序列的损伤; 在未照射的细胞中，遵循修复的时间过程，从而 避免辐射损伤对细胞功能的干扰。 利用宿主细胞再活化方案，残留的DNA链和 碱基损伤将在细胞的碱基序列水平上进行评分， 修复熟练和修复缺陷体质（例如，AT和 XP）。 携带X射线损伤的DNA序列的质粒的拓扑结构必须 在我们关于基因表达的报告中， 在转染的细胞中编码氯霉素乙酰转移酶 pSV2CAT 关于质粒形式与 现在可以实现特定序列的修复。 一起 利用X射线灭活基因表达的数据， 提出用环状质粒为研究x射线提供一个模型 影响染色质中的DNA，其中DNA也排列在 闭合回路 转染到AT细胞中的序列中残留的未修复损伤 特别令人感兴趣，因为某些DNA修复缺陷， 据信，在所有死亡的人中， 45岁之前患癌症的几率

Examining x-ray damage and repair of cellular DNA at the base sequence level required obtaining homogeneous populations of DNA sequences from the irradiated cells. We demonstrated the feasibility of this in studies on highly repetitive component alpha DNA sequence prepared from irradiated monkey CV-l cells. The system is being used to evaluate the time course of repair of alpha DNA and the influence of caffeine on repair. We now extend the studies to alpha DNA residing in irradiated plasmids transfected into host cells. This approach permits studies on x-ray damage and repair of homogeneous populations cloned genes. Eventually, it should provide a host cell react- ivation system for detecting and evaluating the activity of individual DNA repair enzymes and the transfected genes which code for them. Plasmids serve as surrogate targets for cellular DNA. Damage to plasmid sequences are verified before transfection; the time course of repair is followed, in unirradiated cells, thereby avoiding interference from radiation damage to cell functions. With the host cell reactivation scheme, residual DNA strand and base damage will be scored at the base sequence level in cells of repair proficient and repair deficient constitution (e.g., AT and XP). The topology of plasmids carrying x-ray damaged DNA sequences must be taken into account, as in our report on expression of genes coding for chloramphenicol acetyltransferase in cells transfected with pSV2CAT. Information on the relationship of plasmid form and the repair of specific sequences can now be attained. Together with data on x-ray inactivation of gene expression, the studies we propose with circular plasmids provide a model for studying x-ray effects on DNA in the chromatin, where the DNA is also arranged in closed loops. Residual unrepaired damage in sequences transfected into AT cells are of special interest because certain DNA repair deficiencies in AT are believed to be present in as many as 5% of all persons dying of cancer before age 45 years.