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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Molecular analysis of in vivo mutations induced by N-ethyl-N-nitrosourea in the autosomal Tk and the X-linked Hprt genes of mouse lymphocytes.

The endogenous, autosomal Tk gene is a potentially useful reporter of in vivo mutation since it may recover a wider range of mutational events than the X-linked Hprt gene or bacterial transgenes. In this study, we characterized mutations produced in the Tk gene of Tk(+/-) mice and compared them with mutations induced in the Hprt gene. Treatment of Tk(+/-) mice with N-ethyl-N-nitrosourea (ENU) resulted in dose-related increases in Tk mutants, as measured by the frequency of 5-bromodeoxyuridine-resistant (BrdUrd(r)) spleen lymphocytes. ENU-induced mutant frequencies in the Hprt gene, determined by measuring 6-thioguanine-resistant (TG(r)) lymphocytes, were similar to the Tk mutant frequencies. Allele-specific PCR of DNA from BrdUrd(r) lymphocyte clones suggested that 35% of clones from mice treated with ENU and 65% of clones from untreated animals had loss of heterozygosity (LOH) of the Tk gene due to deletion of the functional Tk allele. Reverse transcriptase-PCR/sequencing analysis of BrdUrd(r) and TG(r) clones from ENU-treated mice indicated that point mutations in both genes predominantly occurred at A:T basepairs; however, A:T-->G:C transition was the most common mutation in the Tk gene, while A:T-->T:A transversion was the most frequent mutation in the Hprt gene. Substitution at A:T basepairs in the Hprt gene occurred disproportionately with the mutated dT on the nontranscribed DNA strand, while this strand bias for mutation was not seen in the Tk gene. The results indicate that the specificity of ENU-induced point mutation differs between the two endogenous genes and that the autosomal Tk gene of Tk(+/-) mice is capable of recovering mutations caused by LOH. Environ. Mol. Mutagen. 34:30-38, 1999 Published 1999 Wiley-Liss, Inc.[1]


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