POLYMERASE CHAIN REACTION-DIRECTED DNA-SEQUENCING OF BLEOMYCIN-INDUCED NONDELETION-TYPE, 6-THIOGUANINE-RESISTANT MUTANTS IN CHINESE-HAMSTER OVARY CELL DERIVATIVE AS52 - EFFECTS OF AN INHIBITOR AND A MIMIC OF SUPEROXIDE-DISMUTASE

Bleomycin-induced, 6-thioguanine-resistant, ''non deletion'' mutants pretreated with or without either TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,6,6-tetramethylpiperidine-1 -oxyl), a SOD mimic, were analyzed by polymerase chain reaction (PCR)-directed DNA sequencing in a Chinese hamster ovary (CHO) cell derivative, AS52. Among the 23 bleomycin-induced mutants, six have 3-bp 5'-TGA-3' deletions in the region of 366-371, five have single-base deletions, seven have base substitutions, three have insertions, and two have possible translocations. Among the 16 bleomycin-induced mutants pretreated with TRIEN, six have the 5'-TGA-3' deletion (366-371), two have single-base deletions, one has a 13-bp deletion, four have single-base substitutions, one has a double-base substitution, and two have insertions. Among the 17 bleomycin-induced mutants pre-treated with TEMPOL, six have the same TGA deletions, two have single-base deletions, two have single-base insertions, four have single-base substitutions, one mutant has a 12-bp deletion, one has a 13-bp deletion, and one mutant shows no detectable change in its coding region in the DNA sequence. A possible shift from a ROS-mediated mutational spectrum to a spontaneous mutational spectrum by TRIEN further indicates that reactive oxygen species play an important role in bleomycin mutagenesis in mammalian cells. (C) 1994 Wiley-Liss, Inc.