CARCINOGEN-INDUCED DELETION MUTAGENESIS IN V79 CELLS

肿瘤抑制基因的缺失、突变和失活是一种 重要的一步，导致细胞永生、致癌和 转移性肿瘤进展。主动修复DNA损伤有助于避免 突变和癌症的形成。最近的证据表明，DNA 序列、染色体结构和基因组位置都对 DNA修复和突变的特异性和有效性。到目前为止，它 不可能分离这些变量并研究修复和 在一个以上的基因组位置上对一个基因进行突变。几个稳定的GPT 最近建立了转基因V79细胞系，每个细胞系含有一个 位于基因组中唯一位置的gpt基因的整合拷贝 表现出自发的突变，而不是基因丢失的结果 失活。G12细胞系，当用破碎剂处理时，显示出 小菌落突变表型，可能表明形成 多位点缺失延伸到相邻的必需基因，因此 提供一种方便的方式来评分、选择和分析这种类型的 突变。G12细胞加上一个或两个额外的细胞系(选择 基于博莱霉素和EMS诱导的突变频率)将是 进一步研究。这些细胞中的gpt基因将被定位并将其 通过原位杂交确定V79基因组中的位置。这个 将对G12细胞系小克隆突变表型进行研究 确定它是否是大量删除或重新排列的结果 延伸到gpt基因之外。包括一部分的缺失突变 将对gpt基因进行克隆和测序。中的gpt序列 不可逆转的突变体将通过聚合酶链式反应和 基因内缺失和突变分析。紫外线和EMS诱导的细胞损伤修复 还将使用一种新的技术来分析gpt基因，该技术基于 Hanawalt的基因特异性修复试验。修改后的化验方法将测量 紫外法将BrdUrd掺入gpt基因修复斑块中 在量化之前的含BrdUrd序列的光解 DNA采用南方技术。诱导的序列依赖性 基因内的突变将被分析并与链进行比较 修复的特异性。这些研究的结果将提供新的 对染色体定位在DNA突变中的作用的洞察和帮助 来解释碎屑岩的遗传变异性。

Deletion mutagenesis and inactivation of tumor suppressor genes is an important step leading to cellular immortality, carcinogenesis, and metastatic tumor progression. Active repair of DNA lesions helps to avoid the formation of mutations and cancer. Recent evidence indicates that DNA sequence, chromosomal structure, and genomic location all contribute to the specificity and efficiency of DNA repair and mutagenesis. Until now, it was not possible to separate these variables and study the repair and mutagenesis of one gene at more than one genomic site. Several stable gpt+ transgenic V79 cell lines were recently developed each containing one integrated copy of the gpt gene located at a unique site within the genome and exhibiting spontaneous mutagenesis that is not the result of gene loss of inactivation. The g12 cell line, when treated with clastogens, shows a small colony mutant phenotype which may indicate the formation of multilocus deletions extending into an adjacent essential gene and thus provide a convenient way to score, select, and analyze this type of mutation. The g12 cells plus one or two additional cell lines (chosen based on their bleomycin- and EMS-induced mutation frequencies) will be studied further. The gpt gene in these cells will be mapped and its location within the V79 genome determined by in situ hybridization. The small colony mutant phenotype of the g12 cell line will be studied to determine if it is the result of large deletions or rearrangements extending beyond the gpt gene. Deletion mutations that include a portion of the gpt gene will be cloned and sequenced. The gpt sequences in nonrevertable mutants will be amplified by polymerase chain reaction and intragenic deletions and mutations analyzed. UV- and EMS-induced repair of the gpt gene will also be analyzed using a novel technique based on Hanawalt's gene-specific repair assay. The modified assay will measure the incorporation of BrdUrd into repair patches within the gpt gene by UV photolysis of the BrdUrd-containing sequences prior to quantitation of the DNA by the Southern technique. The sequence dependence of induced mutagenesis within the gene will be analyzed and compared with the strand specificity of repair. The results of these studies will provide new insights into the role of chromosomal location in DNA mutagenesis and help to explain the genetic variability of clastogens.