RADIATION--DELAYED MUTATION & INSTABILITY IN HUMAN CELLS

这项工作的目标是确定已知的现象是否 由于延迟突变发生在体外暴露于电离辐射的人类细胞中， 辐射 传统的突变试验评估了 在一个单一的时间快照突变。 如果辐射暴露导致 基因组的持续不稳定性，传统的检测方法可能会低估 体细胞遗传变异的风险。 假设是 测试的是，延迟突变的出现是诱导的结果， 基因组不稳定性 最初的研究将使用一个不朽的， 近二倍体人B淋巴母细胞样细胞系TK6。 随后的研究 将使用正常人淋巴细胞进行。 以确定是否 出现延迟致死突变，连续采样平板接种效率 将进行辐射的细胞。 确定的持久性 这种表型，在最初的后照射后产生的菌落 将平板进行亚克隆，以确定其继代培养 效率 我们还将确定辐射暴露是否会引起 后代中特定基因座突变的持续升高 辐射细胞 待检测的候选位点是次黄嘌呤 磷酸核糖基转移酶（HPRT）基因座，如果可能，还包括胸苷 激酶基因座（tk）和选择VNTR和微卫星重复基因座。 的 在克隆衍生的hprt中发现的DNA结构改变谱 显示延迟突变表型的突变体将使用 Southern印迹技术或基于PCR的测定，以确定它们是否 类似于自发的突变体还是有独特的特征 与不稳定的诱导有关。 一个单独的措施， 基因组的不稳定性，CAD基因扩增的能力，也将被 在选择用于上述研究的细胞上进行。 我们将 进行体细胞杂交实验，以确定 中国人某些受照后代的延迟突变表型 仓鼠卵巢细胞可以被人类染色体抑制。 我们还寻求 为了确定延迟突变是否是一个被调用的过程， 只有当程序性细胞死亡的途径被废除时，人类细胞才会死亡。 这些研究的总体目标是确定辐射是否 暴露诱导人细胞基因组的持续不稳定性， 并披露是否需要不稳定的倾向， 延迟突变表型是明显的。

The goal of the proposed work is to determine whether the phenomenon known as delayed mutation occurs in human cells exposed, in vitro, to ionizing radiation. Traditional mutation assays evaluate the induction of mutations at a single snapshot in time. If radiation exposure induces a persistent instability in the genome, traditional assays may underestimate the risk of heritable alterations in somatic cells. The hypothesis to be tested is that delayed mutations arise as a result of the induction of genomic instability. Initial studies will be performed using an immortal, near-diploid human B-lymphoblastoid cell line, TK6. Subsequent studies will be performed using normal human lymphocytes. To determine whether delayed lethal mutations arise, serial sampling of the plating efficiency of irradiated cells will be performed. To determine the persistence of this phenotype, colonies arising after the initial post-irradiation plating will be subcloned in order to determine their subcultivation efficiency. We will also determine whether radiation exposure induces a persistent elevation in specific locus mutations in the progeny of irradiated cells. The candidate loci to be tested are the hypoxanthine phosphoribosyltransferase (hprt) locus and, where possible, the thymidine kinase locus (tk) and selected VNTR and microsatellite repeat loci. The spectrum of DNA structural alterations found in clonally derived hprt mutants exhibiting the delayed mutation phenotype will be determined using Southern blotting techniques or PCR-based assays to ascertain whether they resemble spontaneous mutants or whether they have a unique signature associated with the induction of instability. A separate measure of genomic instability, the ability to amplify the CAD gene, will also be performed on the cells chosen for the abovementioned studies. We will carry out somatic cell hybridization experiments to determine whether the delayed mutation phenotype in certain irradiated progeny of Chinese hamster ovary cells can be suppressed by human chromosomes. We also seek to determine whether delayed mutation is a process which is invoked in human cells only when the pathway for programmed cell death is abrogated. The overall goal of these studies is to determine whether radiation exposure induces a persistent instability in the genome of human cells, and to disclose whether a predisposition to instability is required for the delayed mutation phenotype to be evident.