Figure 1.

The Nesp transcript is detected in growing oocytes coincident with establishment of methylation of germline DMRs in the Gnas locus. (A) Scheme of the mouse Gnas locus (Plagge and Kelsey 2006), showing the organization of the overlapping, protein-coding transcripts Nesp, Gnasxl, and Gnas and the noncoding Nespas and 1A transcripts. Transcripts expressed from the maternal allele are indicated above the line, from the paternal allele below the line; Gnas exhibits tissue-specific imprinting, with repression of the paternal allele in a subset of tissues. The location of the DMRs is shown by the rows of filled circles on the methylated allele; the Gnas promoter resides in a biallelically unmethylated CGI (open circles). The positions of the PCR products for bisulphite analysis in B and Figures 3 and 4 are indicated by the hatched bars, and the primers for the RT–PCR assays in C and Figure 2 by labeled arrows. (B) Bisulphite sequences of the Nespas/Gnasxl and 1A DMRs in oocytes isolated at postnatal days 5, 10, and 15 and mature metaphase II (MII) oocytes from adult females. Each row represents the CpG sites of an individual sequenced clone, with filled circles depicting methylated CpG sites (missing circles represent CpGs for which sequence was ambiguous). Sequences obtained from two independent bisulphite treatments are indicated by bracketed sets of methylation profiles. Methylation of these DMRs in MII oocytes has been described previously (Liu et al. 2000; Coombes et al. 2003). (C) RT–PCRs for Nesp, Nespas, Gnasxl, 1A, and Gnas transcripts in day 5, 10, 15 oocytes. Co indicates amplification control with cDNA from an E13.5 embryo; H indicates no template control. The left lanes on each gel show a 100-bp marker ladder, with the 100- or 200-bp markers indicated.