Analysis of spontaneous mutations in a chromosomally located HSV-1 thymidine kinase (TK) gene in a human cell line.
We have developed a system for studying spontaneous mutations at a chromosomally located single-copy HSV-1 thymidine kinase (TK) gene in the human 143 TK- cell line. The neo gene, which confers resistance to the antibiotic G418, was placed next to the TK gene for the purpose of screening out gross chromosomal alterations. TK- mutations were selected using the anti-TK nucleotide analogs trifluorothymidine, acyclovir, and DHPG 9-(1,3 dihydroxy-2-propoxymethyl)-guanine either separately, or in combination to eliminate leaky mutations. Analysis of the TK- mutations by Southern blotting revealed that the majority had undetectable alterations of less than 50 base pairs. The results using the methylation-sensitive enzymes HpaII, AvaI, and SmaI suggest that the inactivation of the TK gene was not due to extensive methylation, although specific methylation of a limited number of MspI sites cannot be ruled out. Reversion studies, however, showed that of 16 mutants analyzed, about half had a very high reversion frequency (approximately 10(-2). This suggests that inactivation of the TK gene may have occurred by a variety of mutational events.
D002460:Cell Line; D002472:Cell Transformation, Viral; D002874:Chromosome Mapping; D004262:DNA Restriction Enzymes; D005796:Genes; D006801:Humans; D009154:Mutation; D018139:Simplexvirus; D013937:Thymidine Kinase / Q000172:deficiency / Q000235:genetics*
D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes