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 mutant s that had base pair alterations, or small deletions or insertions <20 bp. The frequencies of mutants yielding RT-PCR products (mRNA) were the same wh en human EJ30 cells were irradiated in G(1) or S (3-4-fold higher for 6 tha n 3 Gy). However, the frequencies of mutants that did not yield RT-PCR prod ucts were <similar to> 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 tone or more e xons not spliced into mRNA), with 64% of them due to 1-17 bp deletions. Als o, the distributions of molecular alterations in exons, acceptor sites, and donor sites for mutants having splice errors (observed in this study and r eported by others) were similar to those reported for mutants not yielding RT-PCR products (isolated from Russian cosmonauts). In addition, we have fo und previously that large deletions which eliminated 1-9 exons were prefere ntially induced in G(1). Therefore, we postulate that the preferential indu ction of mutants not yielding mRNA is due primarily to splice errors that r esult from deletions preferentially induced during GI. These splice errors would then result either in no message or a message that is rapidly degrade d. (C) 2000 Elsevier Science B.V. All rights reserved.