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

X 射线损伤

基本信息

批准号：
3174103
负责人：
ROBERT E BASES
金额：
$ 20.2万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-01-01 至 1992-03-31
项目状态：
已结题

项目摘要

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.

检查 X 射线损伤和细胞 DNA 底部的修复序列水平需要获得同质的 DNA 群体来自受辐射细胞的序列。我们展示了这在高度重复成分阿尔法研究中的可行性从受辐射的猴 CV-1 细胞制备 DNA 序列。这系统被用来评估阿尔法修复的时间过程 DNA 和咖啡因对修复的影响。我们现在将研究扩展到存在于受辐射的 α DNA 中。质粒转染入宿主细胞。这种方法允许同质群体的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- 用于检测和评估活动的激活系统个体DNA修复酶和编码的转染基因对于他们来说。质粒作为细胞 DNA 的替代靶标。转染前验证质粒序列的损伤；这在未受辐射的细胞中遵循修复的时间过程，从而避免辐射干扰对细胞功能的损害。通过宿主细胞再激活方案，残留的DNA链和碱基损伤将在细胞的碱基序列水平上评分修复熟练和修复不足的体质（例如，AT 和 XP）。携带 X 射线损伤 DNA 序列的质粒的拓扑结构必须被考虑在内，就像我们关于基因表达的报告一样编码转染细胞中的氯霉素乙酰转移酶与 pSV2CAT。有关质粒形式和关系的信息现在可以实现特定序列的修复。一起根据基因表达的 X 射线失活数据，我们的研究提出用环状质粒提供研究 X 射线的模型对染色质中 DNA 的影响，其中 DNA 也排列在闭环。转染至 AT 细胞的序列中残留未修复的损伤之所以受到特别关注，是因为某些 DNA 修复缺陷据信，多达 5% 的死亡者存在 AT 45 岁之前罹患癌症。