The mouse
lymphoma assay (MLA) utilizing the Tk gene is widely used to identify chemical
mutagens. The autosomal location of the Tk gene allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, chemically induced point mutation spectra in the Tk gene of mouse
lymphoma cells have not been characterized. In this study, we determined and compared the mutagenicity and mutational spectra of
N-ethyl-N-nitrosourea (ENU) in the Tk and
Hprt genes of mouse
lymphoma cells. Treatment of L5178Y mouse
lymphoma cells with 100 microg/ml ENU induced a Tk mutant frequency of 756 x 10(-6) and an
Hprt mutant frequency of 311 x 10(-6). Sequence analysis of Tk and
Hprt mutant cDNAs showed a similar overall mutation pattern in the two genes with base-pair substitutions accounting for 83% of non-loss of heterozygosity mutations in the Tk gene and 75% of all mutations in the
Hprt gene. The most common point mutation induced by ENU was G:C --> A:T transition (36 and 28% of independent mutations detected in the Tk and
Hprt genes, respectively). The mutation spectra induced by ENU in both the Tk and
Hprt genes were different from the respective patterns produced in mutants from untreated cells. About 9% of Tk and 7% of
Hprt mutations from control cells were in-frame deletions, whereas no such mutations were found among the ENU-induced Tk and
Hprt mutations. Our results indicate that ENU produces a chemical-specific point mutational profile in the Tk gene of mouse
lymphoma cells that is remarkably similar to that found in the X-linked
Hprt gene. This study provides evidence that the MLA can be used not only to detect point
mutagens but also for analysis of mutational spectra.