CARCINOGEN ASSAY USING POL GENE PROMOTER CONSTRUCTS

这项提议是针对基因工程的发展提出的 将使用报告构建体来测量致癌物质的细胞系 通过利用细胞自身对DNA的反应机制来实现潜力 损坏。我们最近对DNA聚合酶三角洲所做的研究表明，a)它是 参与DNA切除修复的关键DNA聚合酶，以及b)其基因 表达是由培养细胞暴露在DNA损伤中诱导的 紫外线照射的形式。因此，这些新的发现提出了一种可能性 PolDelta基因的表达可作为DNA损伤的一个指标 测试各种化学和物理制剂的遗传毒性。 这个目标已经变得可行，因为我们最近克隆了 人DNA聚合酶增量，并分离出1.7kb的5‘上游启动子 区域。拟议的研究将验证这一概念，即 基因启动子的大小可以作为细胞的一个指标。 对DNA损伤的反应，因此可以用作测试系统 评估任何给定的化学或物理物质的致癌潜力 侮辱。 具体目的1.启动子区域的调控元件将是 具有特征的，并特别关注参与响应的人 对细胞DNA的损伤。利用萤火虫荧光素酶构建报告基因 因为报告基因将被用来研究POL三角洲的反应 推动者。 具体目的2.在AIM L获得的基本信息将用于 修改polDelta基因启动子以保持这些元件 负责对DNA损伤剂的反应。基本反应在 通过突变将把毒剂的缺失减少到最低限度 发起人。这部分工作的目标将具体为 针对polDelta启动子的优化，从而使其 对DNA损伤剂的反应高度敏感和特异。 具体目的3.优化后的报告基因构建稳定 转染人培养细胞系(S)。记者的回应 当这些细胞系暴露于DNA损伤剂时，无论是在 将描述化学和物理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.