SHORT TERM MUTAGEN TESTING WITH HUMAN AND MURINE CELLS

短期诱变剂测试系统将使用突变建立在 小鼠TK+/-L5178Y细胞和人HL-60细胞中的APRT基因座。这个 L5178Y系统在短期诱变剂检测中应用广泛，但也有一些 人们对胸苷激酶基因的可靠性表示怀疑。 作为一个测试标记。该菌株现已成为aprt和aprt的杂合子。 APRT-由其衍生的突变体。杂合子可以被区分为 软琼脂中菌落大小的纯合子。初步分析 表明该系统可用于区分缺失突变体 从其他类型的突变事件中。因为这种蛋白质很容易提纯 并克隆出基因，该系统将用于分子分析。这 将使用适当的DNA杂交研究进行 用于表征DNA序列中可能发生的变化的探针。一系列 然后，特征良好的突变体将被用于逆转研究。一个 对Tk-和aprt-from的突变率进行比较 双重杂合子。 已经对人类细胞系进行了高克隆效率的筛选。HL-60，a 人髓系白血病细胞株，在软琼脂中高效克隆。完毕 经诱变后，已在HL-60细胞中分离到100个aprt突变体。 选定的突变体将在aprt蛋白和 DNA将对合适的突变体进行反向突变测试，以便 建立诱变剂检测制度。对突变体的分子分析将 以确定遗传损伤的特征。上述两个测试系统 将与Ames沙门氏菌系统进行比较，使用一系列 已知致突变谱的农药。

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.