Valentine Carrie R, Raney Jessica L, Shaddock Joseph G, Dobrovolsky Vasily N, Delongchamp Robert R
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
Environ Mol Mutagen. 2004;44(2):128-50. doi: 10.1002/em.20043.
Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants.
单爆发分析应用于phiX174转基因小鼠脾淋巴细胞中基因A突变的正向检测，目的是优化用于鉴定体内突变的分析参数。通过两种不同方法计算了体内爆发的截止值变化对诱导突变频率、增加倍数以及对照小鼠和经N-乙基-N-亚硝基脲（ENU）处理的小鼠之间差异的显著性的影响。为了分离体外爆发，将接种密度降低至每等分试样平均少于10个背景突变噬菌斑。来自对照动物的每等分试样贡献少于60个噬菌斑的突变谱与大肠杆菌或培养的转基因phiX174细胞的对照谱没有显著差异。与每等分试样贡献少于60个噬菌斑的突变相比，经ENU处理的动物的每等分试样大于80个噬菌斑的突变爆发之间的突变谱高度不同（P<0.000001），前者符合ENU诱导突变预期的谱。后者的谱也与对照动物和大肠杆菌不同（P<0.000001），表明这种差异是由离体突变引起的。根据本研究中发现的突变，基因A的报告靶位点总数现在为33个。结果支持这样的解释，即与lacI转基因的结果相反，从对照动物和细胞中分离出的基因A中的100%突变体在大肠杆菌中是固定的。我们将这两个基因之间的差异归因于基因A中的突变热点位点以及一个可检验的假设，即lacI转基因的镶嵌噬菌斑检测低估了离体突变体的频率。
