Abstract
The mouse-lymphoma mutagenesis assay detects forward mutations at the heterozygous thymidine kinase ( tk-1) locus in L5178Y tk +/− 3.7.2C cells. This assay of genotoxicity is widely used to quantitate the mutagenic potential of a variety of chemical and physical agents. A Nco I heteromorphism between the tk a − and tk b + alleles allows the use of Southern blotting to broadly detect two major categories of mutations. These consist of deletions of the functional allele, characterized by absence of a 6.3-kb tk-hybridizing band, and apparent point mutations, indistinguishable from wild-type on blots. Rarely, Southern blots reveal a partial deletion of tk b. The variety of lesions recorded at the heterozygous tk-1 locus may be representative of events important in mammalian carcinogenesis and may include a greater range of mutagenic events than can be observed at hemizygous test loci. To further assess the ability of the mouse-lymphoma assay to detect a variety of mutations and to allow identification of point mutations, we have sequenced the entire tk-1 coding region from both tk a − and tk b + alleles of L5178Y 3.7.2.C mouse-lymphoma cells. Sequences were obtained using PCR amplified double-stranded DNA templates prepared from cytoplasmic RNA from the heterozygous cell line. The two alleles were found to differ by a single TA to GC transversion, altering one amino acid in the deduced amino acid sequence. 4 spontaneous mutants were also sequenced and demonstrated a variety of mutations, including a 6-base pair in-frame deletion, a CG to GC transversion upstream of the start codon, a mutant apparently lacking expression of the tk b allele, and a mutant with apparent wild-type coding sequence for both tk a − and tk b + alleles. The diverse nature of the mutants isolated from L5178Y cells suggests that the mouse-lymphoma mutagenesis assay is capable of detecting a number of mutation types, enhancing the utility of the assay in studying the range of genetic lesions important in human disease. The lesions produced are readily analyzed using a combination of Southern blotting and sequence analysis.
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More From: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
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