| Research Abstract |
Akiyama et al., repoted an elevated frequency of 6-thioguanine-resistant (TG^r) mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in T cells of peripheral blood from atomic bomb survivors and a slight, but significant, positive correlation between the frequency of mutation and radiation dose. Southern blot analysis of DNA from TG^r mutant T cells of atomic bomb survivors, gowever, failed to show a significant difference between the control and survivor groups. To investigate further the mutational events, we have developed a method by which mutational sequence alterations at the hprt locus can be determined by the combination of, i) the multiplex polymerase chain reaction (PCR) and ii) the RT-PCR of cDNA and sequencing. The numbers of independent TG^r mutant T cell clones examined were 41 from a control group of 18 individuals who had received less than 0.005 Gy, and 50 from an exposed group of 24 individuals who had received more than 1.5 Gy (mean dose, 2.45 <plus-minus> 0.85 Gy) . Gross structural alterations, which were detected by multiplex PCR as a loss of or shift in hprt exon-containing fragments of genomic DNA,were found in 10-15% of the clones from both groups, thus indicating there was no significant difference between them. The altered sequences in the HPRTcDNAs recovered from both groups were of various types. Similar proportions of base substitutions (-45%) , deletions or insertions (-25%) , and exon skipping (-20%) were found in both groups, indicating that neither the gross structural alterations in the genomic DNA nor sequence alterations in the hprt cDNA of both groups differ significantly. These results confirm our previous observation that A-bomb-induced HPRT-mutant T cells mostly have been eliminated from peripheral blood over the decades that have elapsed since exposure.
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