title: Identification of imprinted loci by methylation-sensitive representational difference analysis: application to mouse distal chromosome 2.
abstract: Imprinted genes are distinguished by different patterns of methylation on their parental alleles, a property by which imprinted loci could be identified 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 inheritance for this region causes imprinting phenotypes whose molecular basis is only partially understood. RDA led to the recovery of multiple differentially methylated HpaII fragments at two major sites of imprinted methylation: paternal-specific methylation at the Nesp locus and maternal-specific methylation at the Gnasxl locus. Nesp and Gnasxl represent oppositely imprinted promoters of the Gnas gene, which encodes the G-protein subunit, Gsalpha. 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 upstream 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 effective at identifying imprinted methylation, and its potential applications to imprinting studies are discussed.
authors.affiliation: Developmental Genetics Programme, The Babraham Institute, Cambridge, United Kingdom. gavin.kelsey@bbsrc.ac.uk
authors.forename: G
authors.initials: G
authors.lastname: Kelsey
authors.forename: D
authors.initials: D
authors.lastname: Bodle
authors.forename: H J
authors.initials: HJ
authors.lastname: Miller
authors.forename: C V
authors.initials: CV
authors.lastname: Beechey
authors.forename: C
authors.initials: C
authors.lastname: Coombes
authors.forename: J
authors.initials: J
authors.lastname: Peters
authors.forename: C M
authors.initials: CM
authors.lastname: Williamson
mesh_terms: D000818:Animals; D002864:Chromogranins; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics; D005819:Genetic Markers; D018392:Genomic Imprinting; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D002864:Chromogranins; D005819:Genetic Markers; D009419:Nerve Tissue Proteins; C406732:GNAS protein, human; C498642:Gnas protein, mouse; D019205:GTP-Binding Protein alpha Subunits, Gs