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

Abstract

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.

7,12-dimethylbenz[a]anthracene-induced mutation in theTk gene ofTk+/? mice: Automated scoring of lymphocyte clones using a fluorescent viability indicator

“…Early observations with these new mouse models indicate that N-ethyl-N-nitrosourea (ENU) treatment induces similar mutant frequencies in the hprt, aprt, and Tk genes [Wijnhoven et al, 1998;Dobrovolsky et al, 1999b]. These results are consistent with ENU producing mutation mainly through base-pair substitution.…”

“…It is unlikely that the AB stain specifically interferes with the expansion of Tk mutants; earlier we were unable to expand Tk mutant clones identified microscopically [Dobrovolsky et al, 1999b]. It is more likely that either the Tkdeficient phenotype or the selection in BrdUrd diminishes the ability of lymphocytes to proliferate beyond forming a clone in 96WPs.…”

“…If there is a folate deficiency, a possible way to invigorate the de novo pathway of nucleotide synthesis in lymphocytes is to increase the availability of methyl group carriers for the mutant cells, possibly through reformulation of the culture medium. Although Tk KO animals are not very healthy, they can live 7-8 months [Dobrovolsky et al, 1999b]. This suggests that the pyrimidine salvage pathway is not absolutely required at the level of the entire organism, and the reason for poor in vitro expansion of BrdUrd-resistant Tk mutants remains unclear.…”

“…The procedures were described previously [Dobrovolsky et al, 1999b], with the exception that DNA sequencing was performed using BigDye terminator chemistry and a model 377 DNA sequencer (Applied Biosystems, Foster City, CA).…”

Genotoxicity of Acrylamide and Glycidamide: A Review of the Studies by HPRT Gene and TK Gene Mutation Assays

“…This is a single copy gene at position Xq26-27 in humans, codes for HPRT, an enzyme in the nucleotide salvage pathway (21). Mutations at this locus are recognized phenotypically by resistance to 8-azaguanine (8-AZA) or 6-thioguanine (6-TG), purine nucleotide analogues that selectively kill wild-type cells or cells with normal HPRT activity (22). Similarly, in the TK mutation test, cells deˆcient in thymidine kinase due to the mutation of TK ＋/-to TK -/-are resistant to the cytotoxic eŠects of the pyrimidine analogue tri‰uorothymidine (TFT).…”