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  • Title: Analysis of spontaneous mutations in a chromosomally located HSV-1 thymidine kinase (TK) gene in a human cell line.
    Author: Goring DR, Gupta K, DuBow MS.
    Journal: Somat Cell Mol Genet; 1987 Jan; 13(1):47-56. PubMed ID: 3027900.
    Abstract:
    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.
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