TRANSCRIPTION EFFECT ON MUTATION IN HUMAN CELLS

这项提议的目标是确定一个基因的转录水平是否 基因会影响该基因的突变。重要的是要了解 导致体细胞突变的因素，因为 突变在致癌过程中的致病作用。的第一个目标 这个项目是为了开发和鉴定一种具有 诱导型胸苷激酶(Tk)基因座。Galer感应系统将是 被利用了。B淋巴母细胞将用GALER基因稳定地转导 将外部雌激素信号转导到酵母的融合构建体 顺式作用Gal4响应型无人机(上游激活站点)。然后是Gal4 响应性UAS将针对内源性的启动子区域 使用包含8kb同源性的基因替换载体的TK基因 UAS侧翼。tk mRNA诱导的大小和时间进程 将由北方分析确定。第二个目标是测量tk。 低转录水平和高转录水平期间的突变频率(MF) 标准的人体细胞体外突变试验。自发辐射和X射线辐射 将在低转录和高转录期间测量诱导的MF‘s 活动。最后，将确定突变的定性性质。 用人tk cdna探针进行Southern杂交分析。

The goal of this proposal is to determine if the transcription level of a gene affects mutation at that locus. It is important to understand factors which contribute to mutation in somatic cells because of the causative role of mutation in the carcinogenic process. The first aim of this project is to develop and characterize a human cell line which has an inducible thymidine kinase (tk) locus. The GalER induction system will be utilized. B-lymphoblastoid cells will be transfected stabley with a GalER fusion construct which transduces an external estrogen signal to yeast cis-acting Gal4 responsive UAS's (upstream activating sites). Then Gal4 responsive UAS's will be targeted to the promoter region of the endogenous tk gene using a gene replacement vector containing 8 kb of homology flanking the UAS's. The magnitude and time course of tk mRNA induction will be determined by northern analysis. The second aim is to measure tk mutant frequency (Mf) during low and high transcription levels using a standard human cell in vitro mutation assay. Both spontaneous and X-ray induced Mf's will be measured during low and high transcriptional activity. Lastly, the qualitative nature of mutations will be determined using Southern blot analysis with the human tk cDNA probe.