MUTATIONAL LESIONS SPECIFIC FOR IONIZING RADIATION

在确定X射线诱导的光谱的实验过程中， 次黄嘌呤鸟嘌呤磷酸核糖转移酶（HPRT）突变 在人类淋巴母细胞样细胞的基因座中，鉴定了一种复杂的突变。 该突变在一个突变位点的下游3个碱基对处有一个6个碱基对的缺失。 碱基对替换 同样的突变出现了三次 独立地。 根据迄今为止检查的突变体数量， 据计算，这种特殊的突变可能构成多达10%的 所有的X射线引起的事件。 然而，这种改变从未被 在500多个其他突变体中观察到， 自发地或在通过其它试剂诱导后。 因此，这 特定突变可能是辐射暴露的独特标记。 它 提出了发展技术，快速和简便的检测， 这种突变，然后验证它在几个系统中的使用，包括 培养的人细胞，来自治疗的癌症患者的外周淋巴细胞 以及来自受辐射小鼠的脾淋巴细胞。 此外，委员会认为， 拟议的工作将寻求确定其他X射线特异性突变， 在体内小鼠模型和癌症患者中。 具体目标1是发展快速检测的分子技术 和定量的这种X射线特异性突变的人口混合 人类细胞的突变体。 将评估两种可能的方法。 第一种是基于聚合酶链反应（PCR）测定，其利用 与突变区特异性杂交的引物。 二是 基于突变序列在变性梯度中的差异迁移 凝胶。 目的2是检查诱导的剂量-反应关系 这种突变在人类淋巴母细胞中的作用 辐射，通过使用目标1中开发的技术。 第三特定 目的是确定这种突变是否在体内小鼠中诱导 突变分析，最近由Skopek博士开发。 如果X光片 小鼠hprt基因中形成突变体， 将审查其归纳。 此外，还将制定一种方法， 寻找X射线在体内特异性诱导的其他突变，基于 用变性梯度凝胶分析混合突变群体 电泳 最后，具体目标4是确定X射线是否 特定的突变被诱导，并持续存在于癌症患者中， 用高剂量的电离辐射治疗 一些何杰金氏病 患者在治疗后多个月表现出持续升高的突变频率， 治疗结束，而其他人有正常的频率。 这两 将比较亚群以及正常健康成人。 突变dna 还将对这些患者进行额外的X射线特异性筛查， 突变，通过使用目标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.