DNA-sequence specificity of mutations at the human thymidine kinase locus.

We have established a system for the study of DNA-sequence specificity at a functionally heterozygous thymidine kinase (tk) locus in a human lymphoblastoid cell line (TK6). Characterization of the parental locus demonstrated that the 2 tk alleles were fortuitously distinguished by differential gene expression. One round of PCR amplification yielded a specific tk cDNA product only for the functional parental allele. Analysis of cDNA from newly mutated alleles which retain substantial levels of expression is thus simplified. Amplification and sequencing of tk genomic sequences was used for analysis of low expression mutants, and in order to distinguish and characterize deletion and splicing mutations. DNA-sequence analysis of the parental locus identified a frameshift in tk exon 4 of the non-functional parental allele, and surprisingly, an exon 7 frameshift mutation in the functional tk allele. This exon 7 frameshift results in a predicted alteration of the final 21 amino acids of the TK protein, and a C-terminal extension of 131 additional amino acids. Since TK6 is phenotypically TK+, we can infer that this major C-terminal modification does not eliminate enzymatic activity. The system was utilized for the analysis of 36 spontaneous TK- mutants. Loss of heterozygosity accounted for 58% of the mutations, 11% were attributable to intragenic deletions, and the remainder involved point mutations, primarily G:C to A:T transitions.