Cloning and characterization of ovine insulin, insulin-like growth factor-I and -II genes

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Virginia Tech


Genes encoding ovine insulin like growth factor-I (oIGF-D), -II (oIGF-II) and insulin were cloned, sequenced and characterized. The olGF-I gene contains six exons spanning greater than 30 kilobases. Class 1 and class 2 oIGF-I transcripts contained exons 1 and 2 alternatively-spliced to exon 3, respectively. A novel oIGF-I exon (W) was located upstream of exon 1 and was found alternatively spliced to exon 3. No in-frame methionine codon was found in exon W and therefore translation is proposed to initiate at the methionine codon present in exon 3. Using primer extension, the ovine transcription initiation sites were mapped and found to be well conserved among mammalian and avian IGF-I genes. Expression of exon 1-, 2- and W-specific transcripts was examined in seven tissues from adult or fetal sheep using a reverse transcription-polymerase chain reaction (RT-PCR) 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 in general showed the highest expression in adult liver. Exon W transcripts were expressed at low levels in all tissues examined.

To confirm that exon W mRNA produced biologically active IGF-I, an exon W containing cDNA was cloned under the control of a glucocorticoid-inducible MMTV promoter (pMMTV-IGF-IW) and transfected into a bovine mammary epithelial cell line (MAC-T). Stable transfectants were induced with a synthetic glucocorticoid to produce secretable IGF-I. Transcript expression of pMMTV-IGF-IW was confirmed by Northern blot analysis and IGF-I was quantified in the medium of growing cells with RIA. Biological activity of the secreted IGF-I was assayed by measuring the incorporation of [³H]-thymidine into DNA of test MAC-T cells. Media harvested from the pMMTV-IGF-IW transfected clones stimulated labeling of MAC-T cells greater than that of conditioned media from MAC-T cells. Thus, biologically active IGF-I was secreted from pMMTV-IGF-IW cells.

The oIGF-II gene is composed of 9 exons that span approximately 25 kilobases. Approximately 750 nucleotides upstream of oJGF-II exon 1 are the three exons of the ovine insulin gene which are transcribed in the same direction as oIGF-II. Four putative promoters direct transcription of six 5’ non-coding exons (1, 3, 4, 5, 6, and 7), which are alternatively spliced to 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 RT-PCR assay, exon | and 3 transcripts were shown to be expressed in adult but not fetal liver. In addition, a novel transcript that contained exon 1 spliced directly to exon 8 was detected in adult liver. Exon 4 transcripts were not detected, whereas exons 5, 6 and 7 transcripts were detected in both fetal and adult liver. Like the human and rodent genes, the regulation of expression of the oIGF-I and oIGF-II genes are under complex control.