title: Deletion and hypermethylation of thymidine kinase gene in V79 Chinese hamster cells resistant to bromodeoxyuridine.
abstract: Previous studies on V79 Chinese hamster cells have shown that bromodeoxyuridine (BrdU) -resistant variants deficient in thymidine kinase (TK) activity arise by a multistep process which is initiated by a random event and progresses gradually during serial culture in the presence of the drug. In order to determine the molecular basis for the loss of TK activity in these cells, the TK gene was isolated from a lambda phage library of genomic V79 DNA, using a fragment of the human TK gene as a probe. One phage isolated contained the entire TK gene in a 15-kb insert, as demonstrated by the ability of the phage DNA to transform Ltk- mouse cells to the TK+ phenotype. Five fragments spanning the entire gene were then subcloned into the plasmid pUC12 for DNA methylation studies. With these probes it was shown by hybridization analysis that the copy number of the TK gene in V79 cells is about four times the copy number in CHO cells and Chinese hamster liver cells. Southern hybridization analysis of the DNA from first-stage variants partially resistant to BrdU indicated that partial resistance was accompanied by deletion of a number of copies of the TK gene in V79 cells. However, the subsequent gradual transition to full BrdU resistance and full loss of TK activity was correlated with a gradual hypermethylation of sites in the 5' region of the TK gene, with no further change in gene copy number.
authors.affiliation: Department of Zoology, University of California, Berkeley 94720.
authors.forename: T L
authors.initials: TL
authors.lastname: Wise
authors.forename: M
authors.initials: M
authors.lastname: Harris
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D001973:Bromodeoxyuridine / Q000494:pharmacology*; D002872:Chromosome Deletion; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D004351:Drug Resistance; D005786:Gene Expression Regulation; D005796:Genes; D008745:Methylation; D015183:Restriction Mapping; D013937:Thymidine Kinase / Q000235:genetics*; D013997:Time Factors
chemical_list: D004247:DNA; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine