Mutations induced in the HPRT gene by X-irradiation during G(1) or S: analysis of base pair alterations, small deletions, and splice errors.
Reverse transcriptase PCR was performed with mRNA obtained from HPRT mutants that had base pair alterations, or small deletions or insertions <20bp. The frequencies of mutants yielding RT-PCR products (mRNA) were the same when human EJ30 cells were irradiated in G(1) or S (3-4-fold higher for 6 than 3Gy). However, the frequencies of mutants that did not yield RT-PCR products were approximately 10-fold higher in the cells irradiated in G(1) than in those irradiated in S. Sequence analysis of RT-PCR products and genomic DNA showed that 40% of the RT-PCR products had splice errors (one or more exons not spliced into mRNA), with 64% of them due to 1-17bp deletions. Also, the distributions of molecular alterations in exons, acceptor sites, and donor sites for mutants having splice errors (observed in this study and reported by others) were similar to those reported for mutants not yielding RT-PCR products (isolated from Russian cosmonauts). In addition, we have found previously that large deletions which eliminated 1-9 exons were preferentially induced in G(1). Therefore, we postulate that the preferential induction of mutants not yielding mRNA is due primarily to splice errors that result from deletions preferentially induced during G(1). These splice errors would then result either in no message or a message that is rapidly degraded.
Leonhardt
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Radiation Oncology Research Laboratory, University of California at San Francisco, 1855 Folsom St. MCB200, San Francisco, CA 94103, USA. leonardt@flash.net
Trinh
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Chu
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Dewey
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D004249:DNA Damage / Q000528:radiation effects; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004307:Dose-Response Relationship, Radiation; D005091:Exons; D016193:G1 Phase / Q000528:radiation effects*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009154:Mutation; D022821:RNA Splice Sites / Q000528:radiation effects; D012333:RNA, Messenger / Q000032:analysis; D011874:Radiometry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000528:radiation effects*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D022821:RNA Splice Sites; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase