Transformation with DNA from 5-azacytidine-reactivated X chromosomes.
It has been shown that 5-azacytidine (5-Aza-Cyd) can reactivate genes on the inactive human X chromosome. It is assumed that the 5-Aza-Cyd acts by causing demethylation of the DNA at specific sites, but this cannot be demonstrated directly without a cloned probe. Instead, we have utilized the technique of DNA-mediated transformation to show that the 5-Aza-Cyd-induced reactivation occurs at the DNA level. DNAs from various mouse-human or hamster-human hybrid cell lines, deficient for mouse or hamster hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8) and varying in whether they contained either an active or inactive human X chromosome, were used in transformation of HPRT- cells. DNA from the active human X chromosome-containing cell lines yielded HPRT+ transformants, whereas DNA from the inactive X chromosome-containing cells lines did not. The inactive X chromosomal DNA was able to transform thymidine kinase-deficient mouse cells, indicating that the DNA solution was normal. These results confirm that inactivation of the X chromosome involves a DNA modification. Furthermore, DNAs from three cell lines with a 5-Aza-Cyd-reactivated X chromosome also transform HPRT- cells, demonstrating that the 5-Aza-Cyd has altered the DNA structure and supporting the idea that methylation plays a role in X chromosome inactivation.
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D002460:Cell Line; D002999:Clone Cells / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D004303:Dosage Compensation, Genetic / Q000187:drug effects*; D005260:Female; D006801:Humans; D006822:Hybrid Cells / Q000187:drug effects; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D013937:Thymidine Kinase / Q000235:genetics; D014170:Transformation, Genetic; D014960:X Chromosome / Q000187:drug effects
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D013937:Thymidine Kinase; D001374:Azacitidine