X-RAY DAMAGE AND REPAIR OF PRIMATE CELL A DNA SEQUENCES

灵长类细胞 A DNA 序列的 X 射线损伤和修复

• 批准号: 3174100
• 负责人: ROBERT E BASES
• 金额: $ 22.92万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1986-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3174100
• 关键词: DNA repair; HeLa cells; X ray; bacteriophage T4; caffeine; complementary DNA; deoxyribonuclease I; gel electrophoresis; hypoxia; ionizing radiation; nucleic acid sequence; oncogenes; radiation dosage; radiation genetics; radiation sensitivity; radiotracer; tissue /cell culture

Knowledge of specific DNA base sequence damage and repair after ionizing radiation can provide clues to the molecular mechanisms by which radiation damage leads to mutation, carcinogenesis, and cell cycle disturbances relevant to radiation therapy. We can now get this kind of information because of advances in DNA base sequencing methods. The methods are readily applied to study of homogeneous DNA populations such as bacteriophage DNA and highly repetitive component Alpha of primate DNA (172 b.p. repeat). Alphoid DNA's are found in all human cells; they account for 25% of African green monkey cell DNA and their base sequences are known. Monkey cells from continuous lines BSC-1 and CV-1 will be irradiated and their Alpha sequences isolated using EcoR1*, Hind III, and Hae 111 endonuleases and agarose and polyacrylamide gel electrophoretic separation. After 5 feet 32p phosphate end-labeling and secondary restriction, the sites of strand scissions will be studied by the Maxam and Gilbert base sequencing method. There is no information yet available on DNA base sequence damage by ionizing radiation in living cells. Neverthesless, the techniques to learn this are available and the experiments are feasible. In vitro radiation studies on purified DNA were recently published; some preliminary base sequence damage has already been characterized. We proposed to learn the specific base sequence changes which are relevant in living cells. X-ray does-responses and the time course of in vivo repair of the base sequence damage will be studied, comparing this with damage induced under anoxic conditions. Synchronized HeLa and monkey cells will be irradiated and their progress through the cycle monitored by flow cytometry. Premutational base sequence changes which are not corrected after radiation can now be identified for the first time. Studies will be made on caffeine promoted escape from 2 arrest in irradiated cells, and on the progress of base sequence damage repair in the presence and absence of caffeine. Comparative studies with interspersed repetitive primate sequences, the Kpnl 1.2kb family, will be made because these newly discovered repetitive sequences are widely distributed; they are transcribed, and their base sequences and location in relation to specific human oncogenes are under study.

电离后特异性DNA碱基序列损伤与修复的认识 辐射可以提供一些线索， 损伤导致突变、致癌和细胞周期紊乱 与放射治疗有关。 我们现在可以得到这样的信息 因为DNA碱基测序方法的进步。 所述方法 容易应用于同质DNA群体的研究， 噬菌体DNA和灵长类DNA的高度重复组分α（172 B. P.重复）。 在所有的人类细胞中都发现了α DNA，它们解释了 25%的非洲绿色猴细胞DNA及其碱基序列已知。 将对来自连续细胞系BSC-1和CV-1的猴细胞进行辐照， 使用EcoR 1 *、Hind III和Hae 111分离其α序列 琼脂糖和聚丙烯酰胺凝胶电泳 分居 在5英尺32 p磷酸末端标记和二级标记后， 限制，链断裂的位点将由Maxam研究， 吉尔伯特碱基测序法。 目前还没有关于 电离辐射对活细胞DNA碱基序列的损伤。 无论如何，学习这一点的技术是可用的，并且 实验是可行的。 纯化DNA的体外辐射研究 最近发表;一些初步的碱基序列损害已经被 表征了 我们提出要了解特定的碱基序列变化 与活细胞相关的蛋白质 X射线剂量反应与基底修复的时间进程 将研究序列损伤，并与 缺氧条件 同步的HeLa细胞和猴细胞将被照射 并通过流式细胞术监测它们在循环中的进展。 辐射后未纠正的突变前碱基序列变化 现在可以第一次被识别。 研究咖啡因对大鼠逃避逮捕的促进作用， 辐射细胞，并在碱基序列损伤修复的进展， 咖啡因的存在和不存在 比较研究， 将产生重复的灵长类序列，Kpnl 1.2kb家族，因为 这些新发现的重复序列分布广泛， 转录，以及它们的碱基序列和位置， 特定的人类致癌基因正在研究中。