CARCINOGEN-INDUCED DELETION MUTAGENESIS IN V79 CELLS

肿瘤抑制基因的缺失突变和失活是一种 导致细胞永生、致癌和 转移性肿瘤进展。 积极修复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有助于 DNA修复和诱变的特异性和效率。 到现在 不可能将这些变量分开并研究修复， 在一个以上的基因组位点处对一个基因进行诱变。 几个稳定的gpt+ 最近开发了转基因V79细胞系，每个细胞系含有一个 位于基因组内独特位点的gpt基因的整合拷贝 并表现出非基因丢失所致的自发突变 失活。 当用断裂剂处理时，g12细胞系显示出 小菌落突变表型，这可能表明形成 多位点缺失延伸到相邻的必需基因， 提供了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另外的细胞系（选择 基于其博来霉素和EMS诱导的突变频率）， 进一步研究。 这些细胞中的gpt基因将被定位， 通过原位杂交确定V79基因组内的位置。 的 将研究G12细胞系的小菌落突变表型， 确定它是否是大删除或重排的结果 延伸到GPT基因之外。 缺失突变包括一部分 将对GPT基因进行克隆和测序。 中的gpt序列 不可逆突变体将通过聚合酶链反应扩增， 分析基因内缺失和突变。 UV和EMS诱导的修复 还将使用基于以下的新技术来分析GPT基因： Hanawalt基因特异性修复试验 改良的测定将测量 通过UV将BrdUrd掺入gpt基因内的修复斑中 在定量含有BrdUrd的序列之前， DNA的Southern技术。 诱导的序列依赖性 将分析基因内的诱变并与链内的诱变进行比较。 修复的特异性。 这些研究结果将提供新的 深入了解染色体定位在DNA诱变中的作用， 来解释染色体断裂素的遗传变异

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.