Ionizing Radiation Mutagenesis

描述（由申请人提供）：本项目将调查的影响， 细胞的遗传背景对自发和 人淋巴母细胞中的辐射诱发突变。最近，一个p53-null 建立了人淋巴母细胞系，结果表明，与某些 p53突变的细胞系中，细胞不是超突变的。 具体目标1。事实上p53-null细胞显然不是超变的， 但又是超重组的，这就导致了自发性的假设， 内源等位基因的诱变和整合载体中的重组， 而不是基因组不稳定性的等价指标。到目前为止，这两个终点 还没有在单细胞系统中进行比较，这是目标1的目标。 一对匹配的载体pHWT和pHWD可以测量重组 将整合到p53野生型细胞中 型、p53突变型和p53缺失型。背景突变频率为2 内源基因位点，X-连锁次黄嘌呤鸟嘌呤磷酸核糖转移酶 （HPRT）和常染色体，杂合子胸苷激酶（TK），将与 在GPT处重组。 具体目标2。假设p53参与双链断裂 (DSB)修复负责其对诱变的影响，通过参与 同源重组（HR）、非同源末端连接（NHEJ）或两者。目的2 是研究诱变的遗传学，通过确定定量和 破坏NHEJ或HR的定性效果。 嵌合体成形术将被用于产生特定基因的靶向敲除 参与DSB修复，包括KU 8 O和XRCC 4破坏NHEJ，以及RAD 51和 当可行时，两个“纯”敲除，其中两个等位基因 是失活的，也是“条件性”敲除，其中野生型等位基因是 将产生在受调控启动子控制下加回的产物。为每个新 将确定品系、自发突变和辐射诱发突变的频率 在HPRT和TK。此外，对于每个新的细胞系，以及对于p53野生型， p53-突变和p53-无效，hprt的辐射诱导突变谱 并且将确定TK。

DESCRIPTION (provided by applicant):This project will investigate effects of the genetic background of the cell on mechanisms of spontaneous and radiation-induced mutagenesis in human lymphoblast cells. Recently, a p53-null human lymphoblast line was created and it was shown that, unlike in certain p53-mutated lines, the cells are not hypermutable. Specific Aim 1. The fact that p53-null cells are apparently not hypermutable, but are hyperrecombinational, has led to the hypothesis that spontaneous mutagenesis at endogenous alleles and recombination in integrated vectors are not equivalent indicators of genomic instability. To date, these two endpoints have not been compared in a single cell system, and that is the goal of Aim 1. A matched pair of vectors called pHWT and pHWD that can measure recombination at the bacterial gpt locus will be integrated into cells that are p53 wild type, p53-mutated and p53-null. Background mutation frequencies at two endogenous gene loci, X-linked hypoxanthine guanine phosphoribosyltransferase (hprt) and autosomal, heterozygous thymidine kinase (tk), will be compared with recombination at gpt. Specific Aim 2. It is hypothesized that p53 involvement in double strand break (DSB) repair is responsible for its effects on mutagenesis, via involvement in homologous recombination (HR), non-homologous endjoining (NHEJ), or both. Aim 2 is to study the genetics of mutagenesis, by determining the quantitative and qualitative effects of disrupting NHEJ or HR. The relatively new technique of chimeraplasty will be used to create targeted knockouts of specific genes involved in DSB repair, including KU8O and XRCC4 to disrupt NHEJ, and RAD51 and RAD54 to disrupt HR. When feasible, both "pure" knockouts in which both alleles are inactivated and also "conditional" knockouts where the wild type allele is added back under control of a regulated promoter will be created. For each new line, spontaneous and radiation-induced mutation frequencies will be determined at hprt and tk. Also, for each new cell line, and also for p53 wild type, p53-mutated, and p53-null, the radiation-induced mutational spectra for hprt and tk will be determined.