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Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells
M L Applegate1, M M Moore, C B Broder
1Department of Biological Science, Florida State University, Tallahassee 32306.
Abstract:
The mouse lymphoma L5178Y TK+/- 3.7.2C cell line allows quantitation of induced TK(+/-)----TK-/- mutations at the heterozygous thymidine kinase (Tk) locus. TK-/- mutant colonies show a bimodal size distribution, reflecting a difference in the growth rates of the two size classes that is hypothesized to result from different degrees of genetic damage. The two homologous chromosomes 11 containing the alleles of the Tk gene in L5178Y 3.7.2C TK+/- cells are distinguishable at the cytogenetic level. We find, in addition, that the two alleles are distinguishable at the molecular level because of an Nco I restriction fragment length polymorphism at the 3' end of the gene. In a set of 51 large-colony and 48 small-colony TK-/- mutants induced by ionizing radiation or by chemical mutagens, we find that 78, including all except one of the small-colony mutants, have lost the Tk+ allele and that some of these have two to four copies of the remaining Tk- allele. Nineteen of the large-colony TK-/- mutants that do not show Tk+ allele loss show no other structural changes detectable at the level of Southern blot analysis. One shows a partial deletion. The variety of mutagenic lesions recorded at the heterozygous Tk locus may be representative of events observed in human malignancy and may include a wider range of mutagenic events than can be observed at hemizygous test loci.
Insights
The L5178Y TK+/- 3.7.2C cell line quantifies mutations at the thymidine kinase (Tk) locus. Most TK-/- mutants lose the Tk+ allele, with some showing multiple Tk- copies, indicating diverse genetic damage.
Area of Science:
- Genetics
- Molecular Biology
- Toxicology
Background:
- The L5178Y TK+/- 3.7.2C cell line is a model for studying mutations at the heterozygous thymidine kinase (Tk) locus.
- TK-/- mutant colonies exhibit bimodal size distribution, suggesting varying genetic damage levels.
- Homologous chromosomes 11 carrying Tk alleles are distinguishable cytogenetically and molecularly via Nco I RFLP.
Purpose of the Study:
- To characterize the molecular basis of TK-/- mutations induced by ionizing radiation and chemical mutagens in the L5178Y TK+/- 3.7.2C cell line.
- To investigate the genetic alterations at the heterozygous Tk locus.
- To compare mutagenic events at a heterozygous locus with those at hemizygous loci.
Main Methods:
- Utilized the L5178Y TK+/- 3.7.2C mouse lymphoma cell line.
- Induced TK-/- mutations using ionizing radiation and chemical mutagens.
- Analyzed TK-/- mutants using Southern blot analysis to detect allele loss and structural changes.
Main Results:
- Out of 99 TK-/- mutants, 78 showed Tk+ allele loss; most small-colony mutants and some large-colony mutants exhibited this.
- Some mutants had multiple copies of the Tk- allele.
- Nineteen large-colony mutants without Tk+ allele loss showed no detectable structural changes, with one exhibiting a partial deletion.
Conclusions:
- The heterozygous Tk locus in L5178Y cells reveals diverse mutagenic lesions, including allele loss and copy number variations.
- These findings may represent events relevant to human malignancy.
- The heterozygous Tk locus offers a broader spectrum of detectable mutagenic events compared to hemizygous loci.