IONIZING RADIATION MUTAGENESIS

之前关于人体细胞X射线诱变的工作是用TK6完成的 淋巴母细胞。来自同一男性捐赠者的WTK1细胞 抵抗X射线的毒性影响，对其更敏感 诱变作用，在催化分子间作用方面优于TK6 DNA重组。因此，重组修复系统显然 WTK1的功能比TK6的功能更好。这些细胞系提供了 评估重组基因遗传后果的机会 修理。自发突变和X射线诱变的分子性质 来自这两个品系的基因将在X连锁的HPRT基因座进行比较。过剩 WTK1基因突变可能是染色体内缺失 重组，或基因导致的多个点突变 从伪基因转换而来。这些可能性将被区分开来 通过比较突变光谱。每个频谱将由60个 突变体，其特征是：(I)缺失的比例及其程度 在HPRT周围的2MB区域内，(Ii)HPRT mRNA的大小和数量，以及 (3)点突变和部分缺失断裂点的DNA序列。 或者，X染色体和染色体之间的非同源重组 常染色体位点可产生HPRT突变。染色体涂抹- 将使用荧光原位杂交(FISH)来确定 WTK1比TK6诱导更多的X射线诱导的易位。鱼 还将指示是否有任何个体突变是由 X染色体易位。 TK6和WTK1表现出一种“适应性反应”：即低X射线剂量 降低后一种的致突变性。有更大的保护作用 TK6，因此适应可能在很大程度上保护免受诱变过程的影响 不是由重组修复介导的。为了进一步调查这一点， 将测定适应后诱变的突变体的光谱。 二甲基亚砜(DMSO)保护TK6细胞免受X射线的毒性， 但对诱变频率无影响。令人惊讶的是，在那里 是突变谱中的一个相当大的转变，而且它是 假设这是两个相互竞争的过程造成的。在WTK1中， 有一种对诱导突变频率的保护作用，它是 假设DMSO消除通过重组而产生的突变。至 进一步研究这一点，突变谱将在 二甲基亚砜中的辐照。 最后两个目标将探索造成差异的可能原因。 WTK1和TK6的诱变反应。P53的状态将被确定 因为它参与了对DNA损伤的反应，而且有 染色体17p上WTK1和TK6的差异。这两条线路都将是 测序，如果有不同的基因类型，显性阴性的p53 会转入野生型细胞，并将野生型p53转入 缺乏细胞。对辐射毒性和致突变性的影响 将会被确定。如果在HPRT中看到数量差异 焦点在转染体中，X射线诱导的突变光谱将被 特色化的。最后，为了研究细胞凋亡是否参与了 细胞系中的差异性突变。TK6和WTK1将是 Bc1-2基因转染组。如果以这种方式辐射诱导 细胞凋亡可以被调节，然后存活和突变的剂量反应 将进行研究并确定突变谱。

Previous work on X-ray-mutagenesis in human cells was done with TK6 lymphoblasts. WTK1 cells, derived from the same male donor, are more resistant to the toxic effects of X-rays, more sensitive to their mutagenic effect, and are better than TK6 at catalyzing intermolecular DNA recombination. Thus a recombinational repair system apparently functions better in WTK1 than in TK6. These cell lines afford an opportunity to evaluate the genetic consequences of recombinational repair. The molecular natures of spontaneous and X-ray-induced mutants from these two lines will be compared at the X-linked hprt locus. Excess mutants in WTK1 may be deletions arising by intrachromosomal recombination, or multiple point mutations resulting from gene conversions from a pseudogene. These possibilities will be distinguished by comparing mutational spectra. Each spectrum will consist of 60 mutants, characterized as to (i) proportion of deletions and their extent within a 2 MB region around hprt, (ii) size and amount of hprt mRNA, and (iii) DNA sequence of point mutants and partial deletion breakpoints. Alternatively, non-homologous recombination between the X-chromosome and autosomal sites could produce hprt mutants. Chromosome painting- fluorescence in situ hybridization (FISH) will be used to determine if more X-ray-induced translocations are induced in WTK1 than in TK6. FISH also will indicate if any individual mutants resulted from a translocation of the X chromosome. TK6 and WTK1 exhibit an 'adaptive response': i.e., a low X-ray dose reduces the mutagenicity of a later one. There is greater protection in TK6, and so adaptation may protect largely against the mutagenic process not mediated by recombinational repair. To investigate this further, the spectra of mutants induced after adaptation will be determined. Dimethylsulfoxide (DMSO) protects TK6 cells from the toxicity of X-rays, but has not effect on the induced mutant frequency. Surprisingly, there is a considerable shift in the mutational spectrum, and it is hypothesized that two competing processes account for this. In WTK1, there is a protective effect against induced mutant frequency,and it is hypothesized that DMSO eliminates mutants arising via recombination. To investigate this further, mutational spectra will be determined after irradiation in DMSO. The final two aims will explore possible reasons for the differential mutagenic response in WTK1 and TK6. The status of p53 will be determined because it is involved in response to DNA damage, and there are differences between WTK1 and TK6 on chromosome 17p. Both lines will be sequenced and if there are different genotypes, a dominant-negative p53 will be transferred into the wild-type cell, and a wild-type p53 into the deficient cell. Resulting effects on radiation toxicity and mutagenicity will be determined. If quantitative differences are seen at the hprt focus in the transfectants, the X-ray-induced mutational spectra will be characterized. Finally, to investigate whether apoptosis is involved in differential mutability in the cell lines. TK6 and WTK1 will be transfected with the bcl-2 gene. If in this way radiation-induced apoptosis can be modulated, then survival and mutational dose-response studies will be conducted and mutational spectra characterized.