Primary nucleotide structure of predominant and alternate splice forms of equine insulin-like growth factor I and their gene expression patterns in tissues.

This research focused on determining the primary nucleotide sequence of equine insulin-like growth factor I (IGF-I) and observing the gene’s expression patterns in various horse tissues. The findings showed that the equine IGF-I sequence is similar to various other species, and its expression was more abundant in younger horses.

Research Procedure

• The researchers conducted their study on horses of varying ages.
• Total RNA was extracted from the selected tissue samples and thoroughly purified.
• A technique called reverse transcription was employed to produce complementary DNA (cDNA) from the purified RNA. This was followed by amplification using polymerase chain reaction (PCR) to produce sufficient quantities for subsequent steps.
• The cDNA was then subcloned to plasmid vectors. This allowed the researchers to determine the sequence for later comparison with other species.
• Lastly, RNA from various tissue samples was matched with radiolabeled cDNA or complimentary RNA constructs to determine IGF-I gene expression patterns through techniques such as northern blotting and PCR.

Research Findings

• The nucleotide sequence of equine IGF-I was found to be 90% akin to cows’ sequence, 88% similar to humans’ and sheep’s, and 77% similar to rats’.
• In terms of amino acid sequences, the equine IGF-I was identical to those of humans, cows, dogs, and pigs.
• Alternate splicing of IGF-I was noted, with a large PCR product, known as IGF-IB, found to retain the IGF-I exon 4 sequence—similar to rats and mice.
• Multiple IGF-I transcripts were revealed through Northern blot analysis, with predominant sizes being 1.6 and 4.5 kb.
• The researchers found that IGF-I expression was usually detected in organs like the liver, kidney, and cartilage of young foals, but this expression diminished in the cartilage of a 12-month-old horse.

Conclusion and Interpretation

• The nucleotide sequences of equine prepropeptides were different from other species, yet the sequence encoding the mature IGF-I peptide exhibited higher homology.
• The IGF-IB form (the result of alternate splicing) exhibited substantial differences in its carboxy-terminal.
• The researchers speculated that the cleaved terminal’s biological function might be autocrine feedback.
• The IGF-I gene showed notable expression in various tissues, more so in the cartilage, and generally greater in immature horses.