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

Bleomycin-induced, 6-thioguanine-resistant, ''non deletion'' mutants p retreated with or without either TRIEN (triethylenetetramine), a super oxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,6,6-tetramethy lpiperidine-1 -oxyl), a SOD mimic, were analyzed by polymerase chain r eaction (PCR)-directed DNA sequencing in a Chinese hamster ovary (CHO) cell derivative, AS52. Among the 23 bleomycin-induced mutants, six ha ve 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 (366-37 1), two have single-base deletions, one has a 13-bp deletion, four hav e single-base substitutions, one has a double-base substitution, and t wo have insertions. Among the 17 bleomycin-induced mutants pre-treated with TEMPOL, six have the same TGA deletions, two have single-base de letions, two have single-base insertions, four have single-base substi tutions, one mutant has a 12-bp deletion, one has a 13-bp deletion, an d one mutant shows no detectable change in its coding region in the DN A sequence. A possible shift from a ROS-mediated mutational spectrum t o a spontaneous mutational spectrum by TRIEN further indicates that re active oxygen species play an important role in bleomycin mutagenesis in mammalian cells. (C) 1994 Wiley-Liss, Inc.