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: 3174101
• 负责人: ROBERT E BASES
• 金额: $ 23.76万
• 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 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3174101
• 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.

特定 DNA 碱基序列损伤和电离后修复的知识 辐射可以为辐射的分子机制提供线索 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 由于 DNA 碱基测序方法的进步。 方法有 很容易应用于同质 DNA 群体的研究，例如 噬菌体 DNA 和灵长类 DNA 的高度重复成分 Alpha (172 血压重复）。 Alphoid DNA 存在于所有人类细胞中；他们占 25%的非洲绿猴细胞DNA及其碱基序列是已知的。 来自连续系 BSC-1 和 CV-1 的猴细胞将被照射并 使用 EcoR1*、Hind III 和 Hae 111 分离其 Alpha 序列 内切核酸酶和琼脂糖和聚丙烯酰胺凝胶电泳 分离。 经过5英尺32p磷酸末端标记和二次处理后 限制，链断裂的位点将由 Maxam 和 吉尔伯特碱基测序方法。 目前尚无相关信息 活细胞中电离辐射造成的 DNA 碱基序列损伤。 尽管如此，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坏已经被修复 特点。 我们提出学习具体的碱基序列变化 与活细胞相关。 X射线剂量反应和基底体内修复的时间过程 将研究序列损伤，并将其与以下引起的损伤进行比较 缺氧条件。 HeLa 和猴细胞将同步接受照射 以及流式细胞仪监测的循环进展情况。 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。 将对咖啡因促进两起逮捕事件的逃脱进行研究 辐照细胞的碱基序列损伤修复研究进展 咖啡因的存在和不存在。 穿插比较研究 将制作重复的灵长类序列，Kpnl 1.2kb 家族，因为 这些新发现的重复序列分布广泛；他们 被转录，并且它们的碱基序列和位置与 特定的人类致癌基因正在研究中。