Dipartimento di Genetica e Microbiologia "A. Buzzati-Traverso", Universitá di Pavia, Via Abbiategrasso No 207, 27100, Pavia, Italy.
Cytotechnology. 1995 Jun;19(2):137-42. doi: 10.1007/BF00749768.
A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence of phenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with bothHinfI andHae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion withHae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.
建立了一种基于微卫星直接分析的培养哺乳动物细胞的突变检测方法。带型变化反映了从单个碱基改变到复杂重排的 DNA 改变。通过 DNA 指纹图谱，可以同时检查人类基因组中大量常染色体基因座，从而在没有表型选择的情况下最小化要评分的细胞集落样本的大小。对于突变检测，用亚硝胍处理中国仓鼠细胞（V79），并分离和扩增 14 个独立的克隆。从每个克隆提取的 DNA 用 HindIII 和 HaeIII 消化后，用 33.15 和 33.6 探针杂交，获得 DNA 指纹图谱。还分析了未经处理的细胞的 12 个克隆。与未经处理的细胞相比，处理过的克隆的带型存在一些差异；用 HaeIII 消化和用 33.15 探针杂交可以检测到最高频率的诱导变体。结果表明，微卫星序列是高突变位点，可以在不进行表型选择的情况下直接在 DNA 水平上监测化学试剂的诱变潜力。此外，用本文描述的方法，可以区分真正的突变和表观突变，如由 DNA 甲基化变化引起的突变。
