Characterization of the sheep ghrelin gene. (A) Identification of the ovine GHRL transcription start sites. 5′ RLM-RACE of abomasum. M = NEB molecular weight marker 100; no TAP = negative TAP control. (B) Partial transcript sequence showing exon 0 and exon 1. GHRL exon 0 is shown as a black box, exon 1 as a grey box, introns as horizontal lines. Transcription start sites in exon 0 are capitalized. Size (bp) are shown above each exon. The translation initiation site of preproghrelin is shown as ATG. (C) Ethidium bromide stained agarose gel showing the expression of ghrelin (305 bp), exon 2-deleted preproghrelin (Δex2 preproghrelin, 191 bp) and exon 2,3-deleted preproghrelin (Δex2,3 preproghrelin, 82 bp) amplified from the abomasum (Lane 3) and white blood cells (Lane 4) of sheep. The exon structure corresponding to each RT-PCR product is depicted by the adjacent boxes, and the positions of RT-PCR primers are indicated by arrows above exons. Lane 1 contains a 100 bp molecular weight marker and Lane 2 the no template control. (D) Predicted translation and alignment of sheep preproghrelin variants. The signal peptide, ghrelin and obestatin are shown as boxes above corresponding coding sequences. Wild-type preproghrelin code for a 27 amino acid (AA) ghrelin peptide, while Δex2 preproghrelin and Δex2,3 preproghrelin code for a truncated 13 AA ghrelin peptide. Obestatin peptide (sequence indicated by a dotted line) is encoded by the C-terminus of the wild-type preproghrelin and Δex2 preproghrelin, whereas Δex2,3 preproghrelin has a unique 45 amino acid carboxyl terminal sequence.