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 locu s demonstrated that the 2 tk alleles were fortuitously distinguished b y differential gene expression. One round of PCR amplification yielded a specific tk cDNA product only for the functional parental allele. A nalysis of cDNA from newly mutated alleles which retain substantial le vels 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 mu tations. DNA-sequence analysis of the parental locus identified a fram eshift in tk exon 4 of the non-functional parental allele, and surpris ingly, an exon 7 frameshift mutation in the functional tk allele. This exon 7 frameshift results in a predicted alteration of the final 21 a mino acids of the TK protein, and a C-terminal extension of 131 additi onal amino acids. Since TK6 is phenotypically TK+, we can infer that t his major C-terminal modification does not eliminate enzymatic activit y. The system was utilized for the analysis of 36 spontaneous TK- muta nts. Loss of heterozygosity accounted for 58% of the mutations, 11% we re attributable to intragenic deletions, and the remainder involved po int mutations, primarily G:C to A:T transitions.