IONIZING RADIATION MUTAGENESIS

描述：这个项目将研究缺失的分子机制 人类细胞中的突变。人们的注意力集中在比较 自发产生的突变的数量产量和质量性质 或由辐射诱导的，在P53基因变异的密切相关的细胞系中 状态。一些证据表明，所有的p53突变在 它们的表型效应。事实上，目前还不清楚是否增加了 突变体中的突变性是由于缺乏功能蛋白或 以较高浓度存在的突变形式保留了某些特定的 活动。 DNA缺失可能是由同源区域之间的重组引起的。一个 HPRT已经被开发出一个额外的、失活的外显子。一旦它成为现实 整合到细胞中，额外外显子的缺失导致mRNA编码 一种功能蛋白质，这样的细胞可以很容易地选择。自克隆以来 额外外显子两侧的位点允许插入感兴趣的序列， 同源基因参与野生型和突变型p53基因的缺失突变 可以系统地探索突变细胞。 特定目标1将确定P53是否对突变行为起积极作用 或者消极的时尚。各种p53突变表现出的假说 对诱变的不同数量效应将进行测试。首先，一个P53 将使用无推进器-NEO方法构建空行。第二, 以了解P53对突变是否有负面影响，即缺乏 P53功能蛋白导致P53缺失细胞的突变性增加， 将确定自发突变率，以及野生型突变 而显性阴性或功能增强的p53突变等位基因将是 转回空细胞。这些线路将用于进行 比较定量诱变研究。 特定目标2将使用微型基因系统来研究序列的影响 关于缺失突变的同源性。首先，向量将使用 正向或反向Alu重复，具有不同程度的同源性。这些 将其导入野生型和突变型p53细胞。第二, 诱变研究将确定人的自发性 缺失/重组以及暴露于空气中后的诱发频率 紫外线或X射线。重组子的分子结构将 被分析以查看是否对发生的删除不那么严格 在p53突变系中的同源序列之间。最后，细胞系 从AIM 1开始，将使用微型发电系统进行检查。

DESCRIPTION: This project will investigate molecular mechanisms of deletion mutagenesis in human cells. Attention is focused on comparing the quantitative yield and qualitative nature of mutations arising spontaneously or induced by radiation, in closely related cell lines that vary in p53 status. Some evidence suggests that all p53 mutations are not identical in their phenotypic effects. In fact, it is unclear whether increased mutability in p53 mutants is due to the absence of functional protein or the presence in higher concentration of a mutant form that retains certain activity. DNA deletions may arise by recombination between homologous regions. An hprt has been developed with an extra, inactivating exon. Once it is integrated into a cell, deletion of the extra exon leads to mRNA coding for a functional protein, and such cells can be selected easily. Since cloning sites flanking the extra exon allow sequences of interest to be inserted, the involvement of homology in deletion mutagenesis in p53 wild-type and mutant cells can be explored systematically. Specific aim 1 will determine whether p53 acts on mutation act in a positive or negative fashion. The hypothesis that various p53 mutations exhibit different quantitative effects on mutagenesis will be tested. First, a p53 null line will be constructed, using a promoterless-neo approach. Second, to see if there is a negative effect of p53 on mutation, i.e., that lack of p53 functional protein leads to increased mutability in the p53 null cells, spontaneous mutation rate will be determined, as will mutation, wild-type and dominant-negative or gain-of function p53 mutant alleles will be transfected back to the null cell. These lines will be used to conduct comparative quantitative mutagenesis studies. Specific aim 2 will use the minigene system to study effects of sequence homology on deletion mutagenesis. First, vectors will be constructed with direct or inverted Alu repeats, with various degrees of homology. These will be transfected into p53 wild-type and mutant cells. Second, mutagenesis studies will determine the spontaneous rate of deletion/recombination, and the induced frequencies after exposure to ultraviolet light or X-rays. The molecular structure of recombinants will be analyzed to see if there is less stringency for deletions occurring between homologous sequences in the p53 mutant line. Finally, cell lines from aim 1 will be examined with the minigene system.