Genomic organisation of sense and natural antisense transcripts associated with the ghrelin gene. A. The natural antisense transcripts are shown in black and the corresponding genomic structure of the ghrelin gene in grey. The previously reported 20 bp exon 0 and extended (188 bp) exon 1 of the ghrelin gene are also shown. Exons are represented as boxes, introns as horizontal lines and sizes (bp) are indicated above each exon. The direction of transcription of each gene is indicated by arrowheads. A tilde symbol (~) in front of the size of some exons indicates that as RT-PCR primers spanned these exons, their exact ends are unknown. The antisense strand ghrelinOS transcripts overlap with the sense exon -1 and 4, but not with the exon 0–3 of the ghrelin gene. The three TSSs resulting in 86, 63 or 28 base bp exon 4* are shown. Note that it has not been determined which TSS(s) the various ghrelinOS variants employ. GhrelinOS1 contains an approximately 880 bp exon -1*a, a 158 bp exon 2*a, a short 68 base pair exon 2** and exon 4*. GhrelinOS1 was demonstrated by experimental procedures (RT-PCR) in this study and is supported by expressed sequence tag (EST) data (EST CF264800). GhrelinOS2 (demonstrated via RT-PCR in our study) is similar to ghrelinOS1, except in this variant exon -1* is ~625 bp long (exon -1*b). GhrelinOS3 (Incyte clone LIFESEQ4072309) is similar to ghrelinOS1, but lacks exon 2** while exon 2* contains a 4 bp 3' extension (exon 2*b). GhrelinOS4 is similar to ghrelinOS3, but contains exon 2*a, while ghrelinOS5 lacks exon 2* and 2** (both splice variants demonstrated in our study). B. Schematic organisation of the intron junctions of ghrelinOS natural antisense mRNA variants ghrelinOS1–5 described in A. All exons are flanked by GT/AG intron junctions (with the exception of ghrelinOS1, 2 and 4 (2*a to -1*a/b which is flanked by GC/AG).