Non-random deletions at the dihydrofolate reductase locus of Chinese hamster ovary cells induced by α-particles simulating radon

Abstract

This study presents the physical characterization of mutants induced in mammalian cells by high linear energy transfer alpha-particle radiation that simulates exposure to radon daughters. Alpha-Particles from accelerated 4He at 150 keV/microns 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 2.3 x 10(-6) at the 1.0 Gy dose, approximately 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 Val135 to Gly in exon 5; carcinogen-induced reversion to a DHFR+ phenotype at a frequency of 3 x 10(-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 approximately 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 gamma-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 alpha-particles induce double-strand breaks in accessible chromatin loops.

Defined Sequence Modules and an Architectural Element Cooperate To Promote Initiation at an Ectopic Mammalian Chromosomal Replication Origin

“…2B). These stuffer replacement mutants were transfected stably into ectopic sites in hamster DR12 cells that lack the entire DHFR locus (37). The initiation activity of the stuffer replacement mutants in uncloned cell pools was then measured using a PCR-based nascent-strand abundance assay and compared to the activity of the ectopic wild-type ori-␤ fragment (pMCD) and the previously described deletion mutants pMCD⌬AT and pAKO ( Fig.…”

The Dihydrofolate Reductase Origin of Replication Does Not Contain Any Nonredundant Genetic Elements Required for Origin Activity

“…The DR-8A7 cell line is a DHFRdeficient variant of the hemizygous CHO cell line UA21 (57) and has a deletion of the 14-kb region extending downstream from approximately the center of the last intron of the DHFR gene into the intergenic zone (mp 20 to 34) (Fig. 1A) (35). DR-8A7 cells were propagated on minimal essential medium (MEM) supplemented with nonessential amino acids, 100 M hypoxanthine, 16 M thymidine (57), and 10% fetal clone II (HyClone) and were maintained in an atmosphere of 5% CO 2 .…”

“…The vertical marks on the linear axis correspond to initiation sites identified by 2-D gel analysis (23). (B) The DHFR-deficient variant, DR-8A7, which has only one copy of the DHFR locus and has suffered a radon-induced 14-kb deletion encompassing the 3Ј end of the DHFR gene (35). The resulting deletion junction fragments are indicated above and below the map (compare with Fig.…”

The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

“…In Figure 7A and B, the 2D gel results for ori-␤ in the promoterless ⌬Pml-lox cell line are compared with DR-8, a hemizygous DHFR-deficient variant lacking the 3Ј-end of the DHFR gene (Jin et al 1995). In DR-8, there is almost a total lack of replication intermediates at 90 min, both at ori-␤ and in the body of the DHFR gene, regardless of film exposure times or amounts of material loaded onto the gels (Kalejta et al 1998;L.D.…”