SHORT TERM MUTAGEN TESTING WITH HUMAN AND MURINE CELLS

人类和鼠类细胞的短期诱变剂测试

• 批准号: 3250841
• 负责人: MILTON W TAYLOR
• 金额: $ 11.88万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3250841
• 关键词: DNA; chemical carcinogen; gel electrophoresis; genetic mapping; genetic markers; genotype; heterozygote; homozygote; mutagen testing; mutant; pesticides; tissue /cell culture; toxicant screening

Short-term mutagen test systems will be established using mutations at the APRT locus in mouse TK+/-L5178Y cells and in human cell line HL-60. The L5178Y system is extensively used in short-term mutagen testing, but some doubt has been expressed as to the reliability of the thymidine kinase gene as a test marker. This strain has now been made heterozygotic for APRT and APRT- mutants derived from it. Heterozygotes can be distinguished for homozygotes on the basis of colony size in soft agar. Preliminary analysis indicates that this system can be used to differentiate a deletion mutant from other types of mutant events. Since the protein is easy to purify and the gene cloned, this system will be used for molecular analysis. This will be carried out using DNA hybridization studies with the appropriate probes to characterize possible alterations in DNA sequences. A series of well-characterized mutants will then be used in reversion studies. A comparison will be made between the rate of mutation to TK- and APRT- from the double heterozygotes. Human cell lines have been screened for high cloning efficiency. Hl-60, a human myelocytic leukemic cell line, clones efficiently in soft agar. Over 100 APRT- mutants have been isolated in HL-60 following mutagenesis. Selected mutants will be characterized at the level of APRT protein and DNA. Suitable mutants will be tested for reverse mutation in order to establish a mutagen test system. A molecular analysis of the mutants will be done to characterize the genetic lesion. The above two test systems will be compared with the Ames Salmonella system using a series of pesticides of known mutagenic spectrum.

短期诱变剂试验系统将使用 小鼠TK+/-L5178 Y细胞和人细胞系HL-60中的APRT基因座。 的 L5178 Y系统广泛用于短期诱变剂测试，但一些 对胸苷激酶基因的可靠性表示怀疑 作为测试标记。 该菌株现在已被制成APRT杂合子， APRT-从它衍生的突变体。杂合子可以区分为 纯合子的基础上的菌落大小在软琼脂。 初步分析 表明该系统可用于区分缺失突变体 其他类型的突变事件。 由于蛋白质易于纯化 该系统将用于分子生物学分析。 这 将使用DNA杂交研究与适当的 探针来表征DNA序列中可能的改变。 一系列 然后将充分表征的突变体用于回复突变研究。 一 将比较TK-和APRT-的突变率， 双杂合子 人类细胞系已经筛选出高克隆效率。 HL-60，a 人粒细胞白血病细胞系，在软琼脂中有效克隆。 超过 在HL-60细胞中经诱变后分离出100个APRT-突变体。 所选突变体将在APRT蛋白水平上表征， DNA. 将测试合适的突变体的回复突变，以 建立诱变剂试验体系。 对突变体的分子分析 来描述遗传损伤的特征。 上述两个测试系统 将与艾姆斯沙门氏菌系统进行比较， 已知致突变谱的农药。