title: The effects of hypoxia and cysteamine on X-ray mutagenesis in human cells. II. hprt mRNA expression and cDNA sequence analysis of induced mutants.
abstract: Mutants at the hprt locus isolated after treatment of cells of the human lymphoblastoid cell line TK6 with X rays were examined by Northern blot and cDNA sequence analysis. In previous work, Southern blot analysis showed that approximately 25% of the mutants isolated from cultures treated with X rays displayed restriction fragment patterns indistinguishable from wild type. In addition, 38 and 48% of the mutants isolated from cultures treated with X rays under two radioprotective conditions, hypoxia or 25 mM cysteamine, respectively, had normal restriction fragment patterns. In the work presented here, Northern blot and DNA sequence analyses were used to characterize these mutants further. Mutants were classified as having normal size and amount of hprt mRNA, reduced amount or undetectable mRNA, or abnormal size message. Mutants that expressed hprt mRNA were sequenced after reverse transcription and PCR amplification. Sequence analysis is reported for 7 mutants from cultures treated in the absence of protection, 11 from cultures treated under hypoxic conditions, and 11 from cultures irradiated in cysteamine. Striking differences among the three treatment groups were not apparent. All types of mutations at both AT and GC base pairs were observed, including transitions, transversions, small deletions, and insertions. Several mutations affected RNA splicing, leading to exon skipping or inclusion of intron sequences in the final message. Approximately half (14/29) of the mutants sequenced had additions or deletions of one to several nucleotides. Also, 3/29 involved tandem DNA base changes (GG-->TT, C-->AA, AA-->CG). These observations are consistent with a mechanism involving the induction of noncoding or synthesis-blocking lesions that result in polymerase slippage or error-prone bypass synthesis. In addition, potential hotspot sites for mutation by X rays were discovered. At one site in exon 3, the same complex mutation, consisting of a G-->T transversion and a nearby six-base deletion, was detected in three independent mutants. Another mutant had a G-->C transversion at the same base, but without the deletion. At another site in exon 8, three mutations occurred at a run of three consecutive cytosines; these included a -C, a -CC, and a C-->AA. Also in exon 8, two mutations (+T, T-->C) occurred at two consecutive thymines.
authors.affiliation: Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115.
authors.forename: C M
authors.initials: CM
authors.lastname: Denault
authors.forename: T R
authors.initials: TR
authors.lastname: Skopek
authors.forename: H L
authors.initials: HL
authors.lastname: Liber
mesh_terms: D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics*; D003543:Cysteamine / Q000494:pharmacology*; D004249:DNA Damage; D018076:DNA, Complementary / Q000737:chemistry*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D014965:X-Rays
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D003543:Cysteamine; D007041:Hypoxanthine Phosphoribosyltransferase