© 1995 Oxford University Press
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Non-random deletions at the dihydrofolate reductase locus of Chinese hamster ovary cells induced by 
-particles simulating radon
Insutute of Cancer Research
1School of Public Health/Division of Environmental Health Sciences, Columbia University 701 West 168th Street, New York, NY 10032, USA
2To whom correspondence should be addressed
This study presents the physical characterization of mutants induced in mammalian cells by high linear energy transfer 
-Particle radiation that simulates exposure to radon daughters. -Particles from accelerated 4He at 150 keV/µm were used to induce 20 Chinese hamster ovary mutants that are deficient in dihydrofolate reductase (DHFR) activity. Parental cells were the hemizygous UA21 line. Cell survival decreased exponentially in response to radiation dose from 0.25 to 1.75 Gy. Mutants were obtained at 1.0 (17/20) and 1.25 Gy (3/20); treatments at 1.50 Gy failed to yield mutants. The induced frequency of mutation was 2.3x10–6 at the 1.0 Gy dose, 
18-fold greater than the spontaneous mutation rate at this locus. DNA of the 20 confirmed null mutants were examined for alterations in the 25 kb DHFR gene by Southern blotting using a mixed probe that scans a continuous 34 kb of sequence. Deletions were the most prevalent induced change (18/20). Of the two point mutants, DNA sequencing showed that one carries a T:A
G:C base substitution that changed Vall35 to Gly in exon 5; carcinogen-induced reversion to a DHFR+ phenotype at a frequency of 3x10–6 confirmed that the other also carried a single base change. The distribution of deletion break sites in the DHFR locus was non-random. In half of the mutants deletion break sites were clustered within a single 9.4 kb DHFR intron. Fine mapping within the gene of 14 mutants by Southern blotting localized 10 distinct break sites to small restriction fragments (<2 kb). Results of this mapping indicated that three unequivocally independent mutants apparently arose by the same deletion and that others shared single break sites. Deletion sizes in these mutants were determined by Southern analysis using six cosmids and two plasmid probes that together span 500 kb of sequence in the region of the DHFR locus. Probing blots with the cosmids defined a maximal deletion size in 15/17 mutants and confirmed that deletions extended <150 kb in 11, a qualitatively different result from that previously obtained after a similar analysis of 
-ray-induced DHFR- mutants. Since deletions were non-randomly distributed, typically less than replicon size and spared regions containing matrix attachment sites, the results suggest a model whereby -particles induce double-strand breaks in accessible chromatin loops.
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