title: 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.
abstract: Bleomycin-induced, 6-thioguanine-resistant, "non deletion" mutants pretreated with or without either TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,2,6,6-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 pretreated 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.
authors.affiliation: Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston 77555-1010.
authors.forename: J
authors.initials: J
authors.lastname: An
authors.forename: A W
authors.initials: AW
authors.lastname: Hsie
mesh_terms: D000818:Animals; D001483:Base Sequence; D001761:Bleomycin / Q000633:toxicity*; D016466:CHO Cells; D006224:Cricetinae; D003497:Cyclic N-Oxides / Q000494:pharmacology*; D004247:DNA; D004252:DNA Mutational Analysis; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010430:Pentosyltransferases / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D013866:Thioguanine / Q000494:pharmacology; D014266:Trientine / Q000494:pharmacology*
chemical_list: D003497:Cyclic N-Oxides; D013113:Spin Labels; D001761:Bleomycin; D004247:DNA; D013482:Superoxide Dismutase; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D013866:Thioguanine; D014266:Trientine; C001803:tempol