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Title: The Gnas imprinting cluster : identification and analysis of candidate regulatory elements
Author: Coombes, C.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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Genomic imprinting is a mechanism that results in the preferential silencing of one copy of a gene according to its parental origin. The distal region of mouse chromosome 2 is subject to genomic imprinting, and contains a complex cluster of imprinted transcripts. These comprise maternally expressed Nesp, paternally expressed Gnasxl, and the predominantly biallelically expressed transcript Gnas (encoding Gas). Uniquely, transcripts originating from oppositely imprinted promoters Nesp and Gnasxl splice onto, and share common downstream exons with Gnas. The cluster additionally contains two paternally expressed non-coding transcripts, Nespas, which runs antisense to Nesp, and Gnas 1A, which also shares exons with Gnas. The identification of regulatory elements at this locus is essential in order to determine the mechanism by which the cluster is imprinted. Physical characterisation of the Nesp-Gnasxl domain demonstrated that the Nesp and Gnasxl exons were situated 15kb apart, and 30kb upstream of Gnas promoter. Both were found to reside within differentially methylated CpG-rich regions, and were associated with direct repeat features. Allele-specific DNaseI hypersensitive sites (HS), mapping within or directly adjacent to these differentially methylated regions (DMRs) were found to associate with the unmethylated active allele in adult mice. In embryonic stem (ES) cells, two clusters of strong paternal-allele specific DNaseI HS were identified at the 5' end of the Gnasxl DMR. One of these clusters coincided with a region of high sequence conservation between mouse and human, and the first exon of antisense transcript Nespas. This region was denoted candidate regulatory region 1 (CRR1). This study additionally attempted to recapitulate imprinting of the human GNAS1 locus using PAC clone RP1-309F20 (156kb insert containing the GNAS1 locus) in a mouse transgenic system. A consistent DNA methylation profile was established across the transgene in the four transgenic lines analysed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available