A sensitive assay for detecting mutations resulting from unequal homologous recombination without phenotypic selection.
Naturally occurring mutations are composed of a large number of mutations of many types that include mutations resulting from unequal homologus recombination between repetitive elements. The present paper describes a sensitive method for detecting such mutations without phenotypic selection. This system utilizes a tandemly arranged Vr repetitive sequence comprising 6000 copies in the mouse genome that is present in the spacer of the ribosomal RNA gene. For HincII digests of FM3A cell DNA, the Vr probe provides 4 major bands of 2.7 kb, 1.7 kb, 1.6 kb and 1.35 kb and several minor bands. Newly induced mutations due to unequal homologous recombination are observed as disappearance of the minor bands and appearance of extra bands. With this method a spontaneous mutation was detected in 14 cell clones randomly isolated after 60 days of continuous growth. Exposure to 4 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine for 2 h revealed 4 mutations in 11 clones examined. Culturing the cells after treatment in the presence of 12-O-tetradecanoylphorbol 13-acetate enhanced the frequency, yielding 6 mutations in 5 clones. The assay can skip phenotypic selection prior to analysis of DNA changes and hence provides a direct method for monitoring mutations resulting from homologous recombination in non-biased cell populations.
Kominami
R
R
First Department of Biochemistry, Niigata University School of Medicine, Japan.
D000818:Animals; D015139:Blotting, Southern; D004275:DNA, Ribosomal / Q000187:drug effects / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D051379:Mice; D008807:Mice, Inbred BALB C; D008938:Mitosis; D009152:Mutagenicity Tests / Q000379:methods*; D010641:Phenotype; D011995:Recombination, Genetic / Q000187:drug effects; D012091:Repetitive Sequences, Nucleic Acid; D012641:Selection, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity
D004275:DNA, Ribosomal; D008769:Methylnitronitrosoguanidine; D013755:Tetradecanoylphorbol Acetate