Sequence analysis of tka(-)-1 and tkb(+)-1 alleles in L5178Y tk+/- mouse-lymphoma cells and spontaneous tk-/- mutants.
The mouse-lymphoma mutagenesis assay detects forward mutations at the heterozygous thymidine kinase (tk-1) locus in L5178Y tk+/- 3.7.2C cells. This assay of genotoxicity is widely used to quantitate the mutagenic potential of a variety of chemical and physical agents. A NcoI heteromorphism between the tka- and tkb+ alleles allows the use of Southern blotting to broadly detect two major categories of mutations. These consist of deletions of the functional allele, characterized by absence of a 6.3-kb tk-hybridizing band, and apparent point mutations, indistinguishable from wild-type on blots. Rarely, Southern blots reveal a partial deletion of tkb. The variety of lesions recorded at the heterozygous tk-1 locus may be representative of events important in mammalian carcinogenesis and may include a greater range of mutagenic events than can be observed at hemizygous test loci. To further assess the ability of the mouse-lymphoma assay to detect a variety of mutations and to allow identification of point mutations, we have sequenced the entire tk-1 coding region from both tka- and tkb+ alleles of L5178Y 3.7.2C mouse-lymphoma cells. Sequences were obtained using PCR amplified double-stranded DNA templates prepared from cytoplasmic RNA from the heterozygous cell line. The two alleles were found to differ by a single TA to GC transversion, altering one amino acid in the deduced amino acid sequence. 4 spontaneous mutants were also sequenced and demonstrated a variety of mutations, including a 6-base pair in-frame deletion, a CG to GC transversion upstream of the start codon, a mutant apparently lacking expression of the tkb allele, and a mutant with apparent wild-type coding sequence for both tka- and tkb+ alleles. The diverse nature of the mutants isolated from L5178Y cells suggests that the mouse-lymphoma mutagenesis assay is capable of detecting a number of mutation types, enhancing the utility of the assay in studying the range of genetic lesions important in human disease. The lesions produced are readily analyzed using a combination of Southern blotting and sequence analysis.
Liechty
M C
MC
Applied Genetics Laboratories, Inc., Melbourne, FL 32901.
Rauchfuss
H S
HS
Lugo
M H
MH
Hozier
J C
JC
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm; D005091:Exons; D006579:Heterozygote; D008223:Lymphoma; D051379:Mice; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009154:Mutation; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D013937:Thymidine Kinase