Studies of methylation along 28 kilobases of X-chromosome DNA, assayed by Southern blot analysis using cloned X-chromosome-specific probes, indicates that XDNA methylation in normal human cells changes with replication, is not correlated with number of X chromosomes or transcriptional activity, and is less stable and more prevalent than when the human X is in the foreign environment of mouse-human hybrid cells. In contrast with observations of others in heteroploid cells, we observed no derepression of the inactive X in clonal populations of normal human fibroblasts treated with 5-azacytidine. This may reflect the differences in stability of the methylation of the human X in a foreign environment. Our observations preclude ubiquitous methylation differences as the molecular basis for X-chromosome inactivation.
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