Activation of the hprt gene on the inactive X chromosome in transformed diploid female Chinese hamster cells.
Treatment with 5-azacytidine, a potent inhibitor of DNA methylation, was used to induce activation of the selectable hprt gene on the inactive X chromosome in a diploid female Chinese hamster cell line. The transformed, stably diploid cell line F3B was selected in media containing the lethal purine analogue 6-thioguanine, to generate a phenotypically HPRT- mutant, F3BT1, of presumed genotype hprt-/hprt(+), where (+) represents the presumably wild-type allele on the inactive X chromosome. Treatment of F3BT1 with 5-azacytidine resulted in phenotypic reversion to HPRT+ at a frequency greater than 10(-3). Similar treatment of 6-thioguanine-resistant control lines derived from male cells, or from CHO (which has no inactive X chromosome), had no effect on the frequency of phenotypic reversion, indicating that activation of the hprt(+) allele, rather than reversion of the hprt- is responsible. This conclusion is substantiated by documentation of the low mutagenic capacity of 5-azacytidine in this system. Proof that the hprt(+) allele can be activated by 5-azacytidine treatment was obtained in somatic cell hybrids in which hprt gene products from the active and inactive X chromosomes could be distinguished by isoelectric focusing. Our results demonstrate that X-linked gene activation associated with generalized DNA demethylation occurs with high frequency in transformed diploid Chinese hamster cells.
Grant
S G
SG
Genetics Department and Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Worton
R G
RG
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002461:Cell Line, Transformed; D006224:Cricetinae; D004171:Diploidy; D004790:Enzyme Induction; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis / Q000235:genetics*; D007525:Isoelectric Focusing; D009154:Mutation; D010641:Phenotype; D015533:Transcriptional Activation; D014960:X Chromosome
D007041:Hypoxanthine Phosphoribosyltransferase; D001374:Azacitidine