Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Jan;87(1):51–55. doi: 10.1073/pnas.87.1.51

Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells.

M L Applegate 1, M M Moore 1, C B Broder 1, A Burrell 1, G Juhn 1, K L Kasweck 1, P F Lin 1, A Wadhams 1, J C Hozier 1
PMCID: PMC53197  PMID: 1967496

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.

Full text

PDF
51

Images in this article

52Image
on p.52
52Image
on p.52
52Image
on p.52
53Image
on p.53
54Image
on p.54
54Image
on p.54

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blazak W. F., Stewart B. E., Galperin I., Allen K. L., Rudd C. J., Mitchell A. D., Caspary W. J. Chromosome analysis of trifluorothymidine-resistant L5178Y mouse lymphoma cell colonies. Environ Mutagen. 1986;8(2):229–240. doi: 10.1002/em.2860080205. [DOI] [PubMed] [Google Scholar]
  2. Carver J. H., Carrano A. V., MacGregor J. T. Genetic effects of the flavonols quercetin, kaempferol, and galangin on Chinese hamster ovary cells in vitro. Mutat Res. 1983 Feb;113(1):45–60. doi: 10.1016/0165-1161(83)90240-6. [DOI] [PubMed] [Google Scholar]
  3. Cavenee W. K., Dryja T. P., Phillips R. A., Benedict W. F., Godbout R., Gallie B. L., Murphree A. L., Strong L. C., White R. L. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. 1983 Oct 27-Nov 2Nature. 305(5937):779–784. doi: 10.1038/305779a0. [DOI] [PubMed] [Google Scholar]
  4. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  5. Clive D., Johnson K. O., Spector J. F., Batson A. G., Brown M. M. Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutat Res. 1979 Jan;59(1):61–108. doi: 10.1016/0027-5107(79)90195-7. [DOI] [PubMed] [Google Scholar]
  6. Czosnek H., Nudel U., Shani M., Barker P. E., Pravtcheva D. D., Ruddle F. H., Yaffe D. The genes coding for the muscle contractile proteins, myosin heavy chain, myosin light chain 2, and skeletal muscle actin are located on three different mouse chromosomes. EMBO J. 1982;1(11):1299–1305. doi: 10.1002/j.1460-2075.1982.tb01314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dozy A. M., Forman E. N., Abuelo D. N., Barsel-Bowers G., Mahoney M. J., Forget B. G., Kan Y. W. Prenatal diagnosis of homozygous alpha-thalassemia. JAMA. 1979 Apr 13;241(15):1610–1612. [PubMed] [Google Scholar]
  8. Evans H. H., Mencl J., Horng M. F., Ricanati M., Sanchez C., Hozier J. Locus specificity in the mutability of L5178Y mouse lymphoma cells: the role of multilocus lesions. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4379–4383. doi: 10.1073/pnas.83.12.4379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geever R. F., Wilson L. B., Nallaseth F. S., Milner P. F., Bittner M., Wilson J. T. Direct identification of sickle cell anemia by blot hybridization. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5081–5085. doi: 10.1073/pnas.78.8.5081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Honey N. K., Shows T. B. The tumor phenotype and the human gene map. Cancer Genet Cytogenet. 1983 Nov;10(3):287–310. doi: 10.1016/0165-4608(83)90058-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hozier J., Sawyer J., Clive D., Moore M. M. Chromosome 11 aberrations in small colony L5178Y TK-/- mutants early in their clonal history. Mutat Res. 1985 Oct;147(5):237–242. doi: 10.1016/0165-1161(85)90064-0. [DOI] [PubMed] [Google Scholar]
  12. Hozier J., Sawyer J., Clive D., Moore M. Cytogenetic distinction between the TK+ and TK- chromosomes in the L5178Y TK+/- 3.7.2C mouse-lymphoma cell line. Mutat Res. 1982 Dec;105(6):451–456. doi: 10.1016/0165-7992(82)90193-2. [DOI] [PubMed] [Google Scholar]
  13. Hozier J., Sawyer J., Moore M., Howard B., Clive D. Cytogenetic analysis of the L5178Y/TK+/- leads to TK-/- mouse lymphoma mutagenesis assay system. Mutat Res. 1981 Nov;84(1):169–181. doi: 10.1016/0027-5107(81)90060-9. [DOI] [PubMed] [Google Scholar]
  14. Kidd V. J., Wallace R. B., Itakura K., Woo S. L. alpha 1-antitrypsin deficiency detection by direct analysis of the mutation in the gene. Nature. 1983 Jul 21;304(5923):230–234. doi: 10.1038/304230a0. [DOI] [PubMed] [Google Scholar]
  15. Knudson A. G., Jr, Meadows A. T., Nichols W. W., Hill R. Chromosomal deletion and retinoblastoma. N Engl J Med. 1976 Nov 11;295(20):1120–1123. doi: 10.1056/NEJM197611112952007. [DOI] [PubMed] [Google Scholar]
  16. Kozak C. A., Ruddle F. H. Assignment of the genes for thymidine kinase and galactokinase to Mus musculus chromosome 11 and the preferential segregation of this chromosome in Chinese hamster/mouse somatic cell hybrids. Somatic Cell Genet. 1977 Mar;3(2):121–133. doi: 10.1007/BF01551809. [DOI] [PubMed] [Google Scholar]
  17. Lin P. F., Lieberman H. B., Yeh D. B., Xu T., Zhao S. Y., Ruddle F. H. Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences. Mol Cell Biol. 1985 Nov;5(11):3149–3156. doi: 10.1128/mcb.5.11.3149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Minty A. J., Caravatti M., Robert B., Cohen A., Daubas P., Weydert A., Gros F., Buckingham M. E. Mouse actin messenger RNAs. Construction and characterization of a recombinant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA. J Biol Chem. 1981 Jan 25;256(2):1008–1014. [PubMed] [Google Scholar]
  19. Moore M. M., Clive D., Howard B. E., Batson A. G., Turner N. T. In situ analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/- mouse lymphoma cells. Mutat Res. 1985 Aug;151(1):147–159. doi: 10.1016/0027-5107(85)90193-9. [DOI] [PubMed] [Google Scholar]
  20. Moore M. M., Clive D., Hozier J. C., Howard B. E., Batson A. G., Turner N. T., Sawyer J. Analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/- mouse lymphoma cells. Mutat Res. 1985 Aug;151(1):161–174. doi: 10.1016/0027-5107(85)90194-0. [DOI] [PubMed] [Google Scholar]
  21. Oberly T. J., Bewsey B. J., Probst G. S. An evaluation of the L5178Y TK+/- mouse lymphoma forward mutation assay using 42 chemicals. Mutat Res. 1984 Feb;125(2):291–306. doi: 10.1016/0027-5107(84)90079-4. [DOI] [PubMed] [Google Scholar]
  22. Rigaud G., Grange T., Pictet R. The use of NaOH as transfer solution of DNA onto nylon membrane decreases the hybridization efficiency. Nucleic Acids Res. 1987 Jan 26;15(2):857–857. doi: 10.1093/nar/15.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Taylor J. D., Wellman S. E., Marzluff W. F. Sequences of four mouse histone H3 genes: implications for evolution of mouse histone genes. J Mol Evol. 1986;23(3):242–249. doi: 10.1007/BF02115580. [DOI] [PubMed] [Google Scholar]
  24. Thacker J. The molecular nature of mutations in cultured mammalian cells: a review. Mutat Res. 1985 Jun-Jul;150(1-2):431–442. doi: 10.1016/0027-5107(85)90140-x. [DOI] [PubMed] [Google Scholar]
  25. Wang Y. Y., Maher V. M., Liskay R. M., McCormick J. J. Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells. Mol Cell Biol. 1988 Jan;8(1):196–202. doi: 10.1128/mcb.8.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yandell D. W., Dryja T. P., Little J. B. Somatic mutations at a heterozygous autosomal locus in human cells occur more frequently by allele loss than by intragenic structural alterations. Somat Cell Mol Genet. 1986 May;12(3):255–263. doi: 10.1007/BF01570784. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES