Initial identification and verification of novel transcribed ghrelin regions using comparative analyses and RT-PCR. A. Mulan sequence conservation profile for the human and murine ghrelin loci. The horizontal axis displays the input sequence. Evolutionary conserved regions (ECRs, > 70% identity; ≥ 99 bp) are depicted as dark red bars above each pairwise alignment. Preproghrelin coding exons 1 to 4 and the non-coding 20 bp exon 0 (blue), intergenic elements (red) and intron sequence (pink) are marked and the vertical axis shows the percent similarity of the murine ghrelin orthologue to the human sequence. Two conserved intergenic regions, 506 and 2.6 kb upstream of exon 1 of the ghrelin gene, can be seen in red. B. Schematic diagram showing the location of RT-PCR primers employed to verify whether the conserved, intergenic regions identified by Mulan were transcribed from the ghrelin gene. A forward primer in the conserved region immediately upstream of the 20 bp exon 0 and a reverse primer in exon 1 amplified a 278 bp PCR fragment (upper panel). A PCR using another exon 1 primer and a forward primer in the conserved region 2.6 kb upstream of exon 1 resulted in a 227 bp PCR fragment (lower panel). The PCRs confirm that the conserved regions predicted by Mulan correspond to ghrelin gene-derived exons. We have termed the conserved regions exon -1 and extended exon 0.