Abstract
We used two-dimensional electrophoresis (2-DE) of DNA to display thousands of DNA fragments as spots on a gel without the use of probes for detection of gene mutations. Using this technique, we assessed mutation rate in mouse germ cells following paternal radiation exposure. We selected B6C3F1 males and JF1 females as parents. We examined DNA samples of 1,007 progeny, 505 derived from sprematoginial cells irradiated with 4-Gy of X-rays and 502 control progeny. We selected 1,190 paternal spots and 1,240 maternal spots for the mutation screening. Analyses of 595,387 paternal spots in the 4-Gy exposed group permitted us to identify six mutations affecting 12 spots in six mice. Among these, five mutations were deletions involved multiple spots; one deletion involved four spots, three deletions each involved two spots and another one involved a single spot. Molecular studies using high-density oligo microarray revealed that all five deletions were quite extensive, 1.9Mb to 13Mb. Another mutation in the exposed group consisted of a small change of 6-bp gain at a microsatellite. In the control group one spontaneous mutation, 2.3-Mb deletion which involved two spots, was identified among the 583,051 paternal spots. Among the 1,228,352 maternal spots, we found two spontaneous mutations, a 34-bp deletion at a microsatellite locus and a 2.5-Mb deletion. Assuming that each spot as a locus, the estimated spontaneous mutation rate is 0.34 x 10-5/locus/generation. The induction rate is estimated as 0.42 x 10-5/locus/Gy (the confidence interval includes 0). Our estimate is considerably lower than the mean induction rate at the 7-locus tests in mice, which is estimated to be 2-3 x 10-5/locus/Gy. The DNA 2-DE scans about 0.2% of the genome, thus we estimate that 1-Gy of radiation on male spermatogonial cells will induce approximately one relatively large deletion mutation per genome (or animal).
