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• W2045894784 abstract "Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple coples and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK− mutants which are the result of plasmid loss. Under these conditions TK− clones were isolated with a frequency of 5·10−6 both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK− derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK− variants. In the TK− derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK− variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of two single copy TK+ cell lines no TK− mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK− again at a 100-fold higher rate than the original TK+ cell line." @default.
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• W2045894784 date "1989-03-01" @default.
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• W2045894784 title "De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells" @default.
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• W2045894784 doi "https://doi.org/10.1016/0167-4781(89)90042-0" @default.
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