Identification of imprinted loci by methylation-sensitive representationaldifference analysis: Application to mouse distal chromosome 2

Imprinted genes are distinguished by different patterns of methylation on t heir parental alleles, a property by which imprinted loci could be identifi ed systematically. Here, representational difference analysis (RDA) is used to clone HpaII fragments with methylation differences on the maternal and paternal copies of distal chromosome (Chr) 2 in the mouse, Uniparental inhe ritance for this region causes imprinting phenotypes whose molecular basis is only partially understood. RDA led to the recovery of multiple different ially methylated HpaII fragments at two major sites of imprinted methylatio n: paternal-specific methylation at the Nesp locus and maternal-specific me thylation at the Gnasxl locus. Nesp and Gnasxl represent oppositely imprint ed promoters of the Gnas gene, which encodes the G-protein subunit, Gs alph a. The organization of the Nesp-Gnasxl-Gnas region was determined: Nesp and Gnasxl were found to be 15 kb apart, and Gnasxl was found to be 30 kb upst ream of Gnas. Sites of imprinted methylation were also detected at the loci for neuronatin on Chr 2 and for M-cadherin on Chr 8, RDA was highly effect ive at identifying imprinted methylation, and its potential applications to imprinting studies are discussed. (C) 1999 Academic Press.