The ovine insulin-like growth factor-II (oIGF-II) gene is comprised of 9 exons that span approximately 25 kb. Approximately 750 nucleotides upstream of oIGF-II exon 1 are the three exons of the ovine insulin gene that are transcribed in the same direction as oIGF-II. The genomic organization and expression of the oIGF-II gene is similar to that of the human IGF-II gene. Four putative promoters direct the transcription of six 5' noncoding exons (1, 3, 4, 5, 6, and 7), which are alternatively spliced to the shared exons 8, 9, and 10. An ovine exon comparable to human exon 2 has not been identified. Multiple transcription initiation sites were identified for exons 1 and 6 by primer extension analysis. Using a reverse transcriptase polymerase chain reaction (RT-PCR) assay, exon 1 and 3 transcripts were shown to be expressed in adult but not fetal liver. In addition, a novel transcript, which contained exon 1 spliced directly to exon 8, was detected in adult liver. Exon 4 transcripts were not detected in either fetal or adult liver, whereas exon 6 and 7 transcripts were detected in both fetal and adult liver. Exon 5 transcripts were also expressed in both fetal and adult liver, which is in contrast to the tumor cell-specific expression of human exon 5. Like the human and rodent genes, the regulation of expression of the oIGF-II gene is under complex control.

Alternative splicing of ovine insulin-like growth factor-I (IGF-I) transcripts generates three different mRNAs. Class 1 and class 2 transcripts contain exons 1 and 2 spliced to exon 3, respectively. A novel IGF-I mRNA containing exon W is spliced to exon 3 and has been located upstream of exon 1. No in-frame methionine codon was present in exon W and therefore translation is proposed to initiate at the methionine codon present in exon 3. Using primer extension, transcription initiation sites were found 179, 336, and 368 nucleotides upstream of exon 1 and 86, 96, 131, and approximately 850 nucleotides upstream of exon 2. The locations of these transcription initiation sites are well conserved among mammalian and avian IGF-I genes. Expression of exon 1-, 2-, and W-specific transcripts was examined in brain, heart, kidney, liver, lung, skeletal muscle, and spleen from adult ewes or 75-day fetal lambs using a reverse transcriptase-polymerase chain reaction assay. Exon 1 transcripts were the most abundant and found in all fetal and adult tissues. Exon 2 transcripts were found in all tissues and were generally expressed the highest in adult liver. Exon W transcripts were also found to be expressed in all tissues examined. Thus, the three alternatively spliced ovine IGF-I transcripts were expressed in a variety of fetal and adult tissues.

We have isolated and characterized lamb liver cDNAs encoding ovine insulin-like growth factor-I (oIGF-I) precursor polypeptide to study IGF-I gene expression in ruminants. Four cDNA clones were sequenced revealing two different exon 1 sequences (designated 1A and 1B) and four different putative poly(A) adenylation sites. cDNAs containing exon 1A or exon 1B encode precursor polypeptides of 138 or 154 amino acids, respectively. A 130-amino-acid peptide is encoded by all cDNAs examined. These precursors include a hydrophobic leader peptide of varying lengths, the 70-amino-acid oIGF-I, and a 35-amino-acid carboxyl terminal extension peptide. The predicted amino acid sequence of the oIGF-I peptide differs from the human, bovine, and porcine IGF-Is at a single amino acid (at position 66, alanine is substituted for proline) and differs from rat and mouse IGF-Is at 4 and 5 positions, respectively. Both the amino- and carboxy-terminal extension peptides showed regions of extensive sequence homology. Ovine IGF-I amino-terminal peptides are 1 amino acid longer than other mammalian IGFs due to the presence of an extra amino acid (glutamine) present at the proposed boundary of exon 1 and exon 2. Northern blot analysis revealed multiple oIGF-I transcripts in a broad band at 800-1,100 nucleotides and other transcripts of higher molecular weight in liver. There was no detectable expression in either spleen or brain.