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MUTANT FREQUENCIES AND LOSS OF HETEROZYGOSITY INDUCED BY N-ETHYL-N-NITROSOUREA (ENU) IN THE THYMIDINE KINASE (TK) GENE OF L5178YTK+/-3.7.2C MOUSE LYMPHOMA CELLS
Citation:
Chen, T., K H. Brock, AND M M. Moore. MUTANT FREQUENCIES AND LOSS OF HETEROZYGOSITY INDUCED BY N-ETHYL-N-NITROSOUREA (ENU) IN THE THYMIDINE KINASE (TK) GENE OF L5178YTK+/-3.7.2C MOUSE LYMPHOMA CELLS. MUTAGENESIS 17(2):105-109, (2001).
Description:
MUTANT FREQUENCIES AND LOSS HETEROZYGOSITY INDUCED BY N-ETHYK-N-NITROSOUREA (ENU) IN THE THYMIDINE KINASE (tk) GENE IF l5178Y/TK+/-3.7.2C MOUSE LYMPHOMA CELLS
N-ethyl-N-nitrosourea (ENU) is a potent monofunctional-ethylating agent that has been found to be mutagenic in a wide variety of organisms from viruses to mammalian germ cells. To elucidate the mutagenicity of ENU at the Tk+/- locus of mouse lymphoma cells and to confirm the ability of the mouse lymphoma assay (MLA) to detect both point mutations and large DNA alterations, Tk+/- L5178Y cells were exposed to different doses of ENU. Treatment of the cells with ENU resulted in a linear dose response with mutant frequencies of up to 16-fold over control. Evaluation of mutant clone size showed that 36% of the 100 mg/ml ENU-induced clones (66% in control) were small colony mutants and 64% (34% in control) were large colony mutants. DNA isolated from mutants in the control culture and the 100 mg/ml ENU treatment group was analyzed for loss of heterozygosity (LOH) using allele-specific PCR. The majority of the small colony mutants, both ENU-treated (97%) and spontaneous (91%) lost the Tk1b allele. The percentage of allele loss in ENU-induced large colony mutants was distinctly different from that of the control. Twenty-three percent of ENU-induced large colony mutants lost their Tk1b alleles, whereas 73% of the large colony mutants from the control culture lost the allele (p < 0.001). Overall, 50% of the Tk mutants from the 100 mg/ml ENU-treated culture (86% in control) showed LOH. Our data indicate that ENU is a potent mutagen to mouse lymphoma cells and that 100 mg/ml of ENU induces equal numbers of point mutations and chromosomal mutations. This study serves to verify that the MLA detects both point mutations and chromosomal mutations.