Detection and cloning of an X-linked locus associated with a Not1 site that is not methylated on mouse inactivated X chromosome by the RLGS-M method

In looking for genes that escape X chromosome inactivation, we scanned the methylation status of genomic DNA from XX, XO, and XY mice using the method of restriction landmark genomic scanning using methylation-sensitive endon uclease. We detected and cloned a candidate locus and identified the Orf1 g ene. Orf1 has sequence similarities to the B2 repetitive element and human CXORF4 (formerly called EXLM1), which escapes X inactivation. The B2 elemen t spans the 3' terminus of the ORF and the 3' UTR of Orf1. The Orf1 gene en compasses 18.5 kb of genomic DNA including II exons and 10 introns. Taking advantage of genomic polymorphisms present between MSM and C3H/He, we showe d that murine Orf1 is mapped to the proximal region of the X chromosome. De spite the unmethylation of the Not1 site, Orf1 is subject to X inactivation . (C) 1999 Academic Press.