MUTATIONAL LESIONS SPECIFIC FOR IONIZING RADIATION

在实验过程中确定X射线诱发的光谱 次黄嘌呤鸟嘌呤磷酸核糖转移酶(HPRT)基因突变 在人类淋巴母细胞中发现了一个复杂的突变。 该突变具有6个碱基对缺失3个碱基对的下游 碱基对替换。同样的突变已经出现了3次 独立的。根据到目前为止检查的突变基因的数量， 计算出这种特殊的突变可能构成多达10%的 HPRT的所有X射线诱发事件。然而，这种变化从来没有发生过 在500多个其他被检查的突变体中观察到 自发地或在被其他药剂诱导后。因此，这 特定的突变可能是辐射暴露的一个独特标志。它 建议开发快速、简便的检测技术。 这种突变，然后验证它在几个系统中的使用，包括 培养中的人类细胞，癌症患者的外周淋巴细胞治疗 和来自受辐射小鼠的脾淋巴细胞。此外， 这项拟议的工作将寻求确定其他X射线特有的突变 在活体小鼠模型和癌症患者中。 具体目标1是开发用于快速检测的分子技术 并在混合型人群中定量检测这种X射线特异性突变 HPRT--人类细胞的突变体。将对两种可能的方法进行评估。 第一种是基于聚合酶链式反应(PCR)的分析，利用 与突变区特异杂交的引物。第二个是 基于变性梯度下突变序列的差异迁移 凝胶。目标2是检查诱导的剂量-反应关系。 对体外培养的人淋巴母细胞的这种突变的影响 辐射，通过使用目标1中开发的技术。第三具体 目的是确定这种突变是否在活体小鼠中诱导 突变测试，最近由斯科佩克博士开发。如果X光特定 在小鼠HPRT基因中形成突变体，剂量-反应关系 它的归纳将被检验。此外，还将开发一种方法来 寻找X光在体内特异性诱导的其他突变，基于 变性梯度凝胶分析混合突变群体 电泳法。最后，具体目标4是确定X射线是否 特定的突变被诱导并持续存在于那些已经 用高剂量的电离辐射治疗的。某些霍奇金氏病 患者在数月后表现出持续的突变频率升高 治疗结束，而其他人的频率正常。这两个 亚组以及正常的健康成年人将被比较。突变DNA 也将对这些患者进行额外的X射线特异性筛查 突变，通过使用在AIM 3中开发的方案。

During the course of experiments to define the spectrum of X-ray-induced mutations at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus in human lymphoblastoid cells, a complex mutation was identified. This mutation has a six base-pair deletion 3 base-pairs downstream of a base-pair substitution. The same mutation has appeared 3 times independently. Based on the number of mutants examined so far, it has been calculated that this particular mutation may constitute as much as 10% of all X-ray-induced events at hprt. However, this alteration has never been observed in more than 500 other mutants examined that arose either spontaneously or after induction by other agents. Consequently, this specific mutation may be a distinctive marker for radiation exposure. It is proposed to develop the technology for the rapid and facile detection of this mutation and then to validate its use in several systems, including human cells in culture, peripheral lymphocytes from cancer patients treated with radiation, and splenic lymphocytes from irradiated mice. Furthermore, the proposed work will seek to identify other X-ray specific mutations in the in vivo mouse model and in the cancer patients. Specific Aim 1 is to develop molecular techniques for the rapid detection and quantification of this X-ray-specific mutation in populations of mixed hprt- mutants of human cells. Two potential approaches will be evaluated. The first is based on a polymerase chain reaction (PCR) assay utilizing a primer which hybridizes specifically to the mutant region. The second is based on differential migration of mutant sequences in denaturing gradient gels. Aim 2 is to examine the dose-response relationship for the induction of this mutation in human lymphoblast cells treated in vitro with radiation, by use of the techniques developed in Aim 1. The third Specific Aim is to determine whether this mutation is induced in an in vivo mouse mutation assay, recently developed by Dr. Skopek. If the X-ray specific mutant is formed in the mouse hprt gene, the dose-response relationship of its induction will be examined. Also, an approach will be developed to look for other mutations specifically induced in vivo by X-rays, based on analyses of mixed mutant populations by denaturing gradient gel electrophoresis. Finally, Specific Aim 4 is to determine whether the X-ray specific mutation is induced and persists in cancer patients who have been treated with high doses of ionizing radiation. Some Hodgkin's disease patients exhibit persistently elevated mutant frequencies many months after the end of treatment, while others have normal frequencies. These two subsets, as well as normal healthy adults, will be compared. Mutant DNAs from these patients also will be screened for additional X-ray specific mutations, by use of the protocols developed in Aim 3.