CARCINOGEN ASSAY USING POL GENE PROMOTER CONSTRUCTS

这项建议是针对发展基因工程 将使用报告构建体来测量致癌性的细胞系 利用细胞自身对DNA的反应机制， 损害我们最近对DNA聚合酶delta的研究表明，a）它是 参与DNA切除修复的关键DNA聚合酶，和B）其基因 通过将培养的细胞暴露于DNA损伤来诱导表达。 紫外线照射的形式。因此，这些新发现提出了一种可能性， pol δ基因表达可用作DNA损伤指标， 测试各种化学和物理制剂的遗传毒性。 自从我们最近克隆了 人DNA聚合酶δ并分离了1.7kb 5 ′上游启动子 地区 拟议中的研究将验证这一概念，即应对措施 pol δ基因启动子的表达可作为细胞凋亡的指标 对DNA损伤的反应，因此可以用作测试系统， 评估任何特定化学或物理物质的致癌潜力 侮辱。 具体目标1.启动子区的调控元件将是 特别关注参与应对行动的人 对细胞DNA的损伤使用萤火虫荧光素酶的报告基因构建体 因为报告基因将用于研究pol δ的反应 启动子 具体目标2.在目标1中获得的基本信息将用于 修饰pol δ基因启动子以保持那些元件， 负责对DNA损伤剂的反应。 基础反应， 通过以下诱变将试剂的缺失降至最低 发起人。这部分工作的目标将具体 涉及pol δ启动子的优化， 其对DNA损伤剂的反应高度敏感和特异。 具体目标3.优化的报告基因构建体将稳定表达。 转染到人培养细胞系中。记者的回应 当这些细胞系暴露于DNA损伤剂时， 将表征化学和物理DNA损伤剂的形式。

This proposal is directed toward development of genetically engineered cell lines that will use reporter constructs to measure carcinogenic potential by making use of the cells' own response mechanisms to DNA damage. Our recent work with DNA polymerase delta has shown that a) it is a key DNA polymerase involved in DNA excision repair, and b) that its gene expression is induced by exposure of cultured cells to DNA damage in the form of UV irradiation. Thus, these novel findings raise a possibility that pol delta gene expression may be used as an index of DNA damage for testing the genetic toxicity of a variety of chemical and physical agents. This goal has become feasible since we have recently cloned the gene for human DNA polymerase delta and have isolated a 1.7 kb 5' upstream promoter region. The proposed studies will validate the concept that the response of the pol delta gene promoter can be used as an index of the cellular response to DNA damage, and therefore may be used as a test system to evaluate the carcinogenic potential of any given chemical or physical insults. SPECIFIC AIM 1. The regulatory elements of the promoter region will be characterized, with specific attention to those involved in the response to cellular DNA damage. Reporter gene constructs using firefly luciferase as the reporter gene will be used to study the response of the pol delta promoter. SPECIFIC AIM 2. The basic information obtained in Aim l will be used to modify the pol delta gene promoter to maintain those elements which are responsible for the response to DNA damaging agents. Basal response in the absence of the agents will be reduced to the minimum by mutagenesis of the promoter. The goal of this part of the work will be specifically directed to the optimization of the pol delta promoter so that it is highly sensitive and specific in its response to DNA damaging agents. SPECIFIC AIM 3. The optimized reporter construct will be stably transfected into human cultured cell line(s). The response of the reporter gene when these cell lines are exposed to DNA damaging agents, both in the form of chemical and physical DNA damaging agents will be characterized.