We examined the sequence of DNA synthesis of the human active, inactive and reactivated X chromosomes in mouse-human hybrid cells. The two independent reactivants, induced by 5-azacytidine (5-azaC), expressed human hypoxanthinephosphoribosyl transferase (HPRT), and one also expressed human glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK). Restriction enzyme analysis of DNA methylation at the re-expressed loci revealed hypomethylation of CpG clusters, that characterizes the relevant genes on the active X. The transfer of active and inactive X chromosomes from the native environment of the human fibroblast to the foreign environment of the hybrid cell did not affect the specific replication sequence of either human X chromosome. The silent X chromosome when reactivated, remained allocyclic, and the first bands to replicate were the same as prior to reactivation. In one reactivant, however, further progression of replication was significantly altered with respect to the order in which bands were synthesized. This alteration in the replication of the silent X following 5-azaC-induced reactivation suggests that DNA methylation may modulate the replication kinetics of Chromosomal DNA.
ASJC Scopus subject areas
- Cell Biology