PCR-DIRECTED DNA-SEQUENCING OF NONDELETION HPRT- MUTANTS INDUCED BY BLEOMYCIN IN CHO K1-BH4 CELLS

Bleomycin is one of the radiomimetic antibiotics which induces DNA dou ble-strand breaks by highly specific free radical attack on deoxyribos e moieties in DNA. Earlier, we have shown that bleomycin induces a hig h proportion of large deletions involving one or more exons in the hyp oxanthine-guanine phosphoribosyltransferase (hprt) locus in a Chinese hamster ovary (CHO) cell line CHO K1-BH4, in which no spontaneously oc curring large deletions were detected by a polymerase chain reaction ( PCR)-based deletion screening essay. Here we report the molecular natu re of another class of mu to nts in which we did not observe any abnor mal exon pattern. We refer to these mutants as the ''nondeletion'' typ e. Since bleomycin is a reactive oxygen species (ROS)-generating agent , we also studied whether the change of intracellular levels of ROS ma y affect the bleomycin-induced mutation spectra. We therefore also inv estigated the hprt mutation spectra induced by bleomycin with pretreat ment by TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inh ibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic. Analysis of these three bleomycin-induced ''nondeletion'' mutation spectra revealed that 5'-G (T) under bar C-3' or 5'-G (C) und er bar C-3' sequences were the hot spots for single basepair deletions . Other types of mutation include abnormal cDNA or no cDNA amplificati on on the hprt locus. Due to the smell sample size, we are unable to d raw a definitive conclusion about the effects of TRIEN and TEMPOL on b leomycin-induced spectrum of ''nondeletion'' type hprt mutations. Envi ron. Mol. Mutagen. 32:244-250, 1998 (C) 1998 Wiley-Liss, Inc.