Watson A J, Hankinson O
Laboratory of Biomedical and Environmental Sciences, School of Public Health, University of California, Los Angeles 90024.
Carcinogenesis. 1988 Sep;9(9):1581-6. doi: 10.1093/carcin/9.9.1581.
High mol. wt genomic DNA from a genetically dominant aryl hydrocarbon hydroxylase (AHH)-deficient mutant cell line derived from the mouse hepatoma cell line Hepa-1 was used to transfect the parent cell line. AHH-deficient transfectants were recovered following single-step selection in medium containing the carcinogen benzo[a]pyrene. The transfectants arose at a frequency of 2 x 10(-7). This frequency was at least 4-fold greater than the frequency of spontaneous forward mutation in this cell line. In another set of experiments, dominant mutant DNA was co-transfected along with the selectable plasmid pSV2ecogpt into parental Hepa-1 cells. The frequency of co-transfection was determined to be 3 x 10(-8). This frequency was approximately 150 times greater than that expected on the basis of coincident but unrelated spontaneous mutation and plasmid uptake. Both types of transfectants were judged, following somatic cell hybridizations, to possess the dominant phenotype of the mutant cell line, demonstrating that a trans-acting dominant negative regulator of AHH was transferred in these experiments. DNA transfection should therefore provide a means for the molecular cloning of the gene that encodes the dominant regulator.
从小鼠肝癌细胞系Hepa-1衍生的具有遗传显性芳烃羟化酶（AHH）缺陷的突变细胞系中提取的高分子量基因组DNA，用于转染亲代细胞系。在含有致癌物苯并[a]芘的培养基中进行单步选择后，获得了AHH缺陷的转染子。转染子出现的频率为2×10^(-7)。该频率比该细胞系中自发正向突变的频率至少高4倍。在另一组实验中，显性突变DNA与可选择质粒pSV2ecogpt一起共转染到亲代Hepa-1细胞中。共转染的频率被确定为3×10^(-8)。该频率比基于同时发生但不相关的自发突变和质粒摄取所预期的频率大约高150倍。在体细胞杂交后，两种类型的转染子都被判定具有突变细胞系的显性表型，表明在这些实验中转移了AHH的反式作用显性负调节因子。因此，DNA转染应该为编码显性调节因子的基因的分子克隆提供一种手段。
