A MUTATIONAL HOT-SPOT INDUCED BY N-HYDROXY-AMINOFLUORENE IN DIHYDROFOLATE-REDUCTASE MUTANTS OF CHINESE-HAMSTER OVARY CELLS

Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 muM N-hydroxy-aminofluorene in four separate exp eriments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6) . The nature of the induced changes in each of the mutants was determi ned by direct sequencing following polymerase chain reaction amplifica tion, or in one instance, by Southern blot analysis. Nearly all (15/17 ) of the mutations were single base changes. Consistent with the bindi ng specificity of this chemical, all mutations were targeted to guanin e bases. The predominant change was G:C --> T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the ind uced point mutations arose at this position. These results are compare d with our previous analyses of mutants induced with the related aroma tic amine, N-2-acetoxy-2-acetyl-amino-fluorene.