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MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY MUTAGENS IN THE TK GENE OF MOUSE LYMPHOMA CELLS
Chen, T., K H. Brock, AND M M. Moore. MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY MUTAGENS IN THE TK GENE OF MOUSE LYMPHOMA CELLS. Presented at Society of Toxicology, San Francisco, CA, March 25-29, 2001.
MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY BROMATE AND N- ETHYL-N-NITROSOUREA IN THE TK GENE OF MOUSE L YMPHOMA CELLS
The mouse lymphoma assay is widely used to identify chemical mutagens The Tk +1- gene located on an autosome in mouse lymphoma cells may recover a wide range of mutational events such as point mutation, deletion and recombination. In this study, we have identified about 1,000 Tk mutations from control" N-ethyl-N-nitrosourea (ENU)- treated and bromate- treated mouse lymphoma cells by using PCR analysis for loss of heterozygocity (LOH) at the heteromorphic microsatellite within the Tk locus" and R T /PCR and sequence of the Tk coding area. Measurement of mutant clone size showed that 36% of the ENU-induced mutants, 66% of the spontaneous mutants and 90% of the bromate-induced mutants were small clones. LOR analysis revealed that allele loss is common in both large and small clones. The percentage of allele loss in ENU-induced, spontaneous and bromate-induced large colony mutants were 31 %, 67% and 94% respectively. However, the percentages of allele loss in small colony mutants were similar in the three different groups (98% in ENU, 91% in control" and 99% in bromate). Sequence analysis of Tk mutant cDNA demonstrated that ENU induced primarily base pair substitutions (72%) and exon splice mutations (28%). The most common base pair substitution was G:C > T:A transversion. About 30% of mutations were in-frame deletions in the control" whereas no frameshifts were found in ENU-induced Tk mutants. Seventy five percent of ENU-induced Tk base pair substitutions occurred at the mutated guanine and thymidine (possible targets of ENU binding) on the non-transcribed DNA strand, while no such strand-related bias was found in the spontaneous Tk mutants. The results suggest that most of small size Tk mutants result from LOH mutation while large size lK mutants generated via different mechanisms and are mutagen dependent; the LOH, like clone size, of the Tk mutants as well as the mutation spectra could reflect the nature of mutagens.
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Record Details:Record Type: DOCUMENT (PRESENTATION/ABSTRACT)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY
ENVIRONMENTAL CARCINOGENESIS DIVISION
GENETIC AND CELLULAR TOXICOLOGY BRANCH