Fuscoe J C, O'Neill J P, Machanoff R, Hsie A W
Mutat Res. 1982 Sep;96(1):15-30. doi: 10.1016/0027-5107(82)90013-6.
We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10(6) cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 muM) is then added directly to the growing culture and AS-resistant (ASr) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT- clones. The ASr phenotype is characterized both physiologically and biochemically. All ASr clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.
我们描述了一种利用选择性试剂L-重氮丝氨酸（AS）对中国仓鼠卵巢细胞次黄嘌呤-鸟嘌呤磷酸核糖转移酶（hgprt）位点的回复突变进行定量的检测方法。确定了最佳AS浓度、细胞密度和表型表达时间等条件。处理后，将10⁶个细胞的重复培养物在100毫米培养皿中培养48小时以进行表型表达。然后将AS（10 μM）直接添加到正在生长的培养物中，对AS具有抗性（ASr）的细胞形成可见菌落。该检测方法用于定量ICR-191、ICR-170和N-乙基-N-亚硝基脲诱导的独立分离的HGPRT⁻克隆的回复突变。对ASr表型进行了生理和生化特征分析。所有分离出的ASr克隆对AS和氨甲蝶呤均具有稳定抗性，但对6-硫鸟嘌呤敏感。它们还重新表达了HGPRT酶。此外，有几个回复突变体显示含有改变的HGPRT。这些数据进一步证明ICR-191和ICR-170在哺乳动物细胞中引起结构基因突变，也表明ICR-191、ICR-170和N-乙基-N-亚硝基脲在中国仓鼠卵巢细胞中诱导相似类型的突变。
