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

Objective-To isolate, clone, and determine primary nucleotide sequence of e quine insulin-like growth factor I (IGF-I) and to examine IGF-I gene expres sion in tissues and cartilage from horses. Animals-Horses of various ages. Methods-Total RNA was isolated from tissues and purified. Complementary DNA (cDNA) was derived by reverse transcription and polymerase chain reaction (PCR) amplification and subcloned to plasmid vectors for sequencing and com parison with other species. Total RNA from various tissues was probed with radiolabeled cDNA or complimentary RNA constructs by use of northern blotti ng, tube hybridization, or PCR procedures to determine IGF-I expression pat terns. Results-Nucleotide sequence of equine IGF-I was 90% homologous to that of c ows, 88% homologous to that of humans and sheep, and 77% homologous to that of rats. Amino acid sequence was identical to that of humans, cows, dogs, and pigs. A larger PCR product (IGF-IB) was consistent with alternate splic ing with retention of IGF-I exon 4 sequence, similar to rats and mice. Nort hern blot analysis revealed multiple IGF-I transcripts; predominant sizes w ere 1.6 and 4.5 kb. The IGF-I message was commonly detected in liver, kidne y, and cartilage from young foals and was diminished in cartilage from a 12 -month-old horse. Conclusions-Nucleotide sequences of equine pre-propeptides were different f rom those of other species, but the sequence coding the mature IGF-I peptid e was more closely homologous. The larger IGF-IB form differed substantiall y in the carboxy-terminal. The biological action of the cleaved terminal wa s speculated to be autocrine feedback. Expression of IGF-I was apparent in many tissues, including cartilage, and was greater in immature horses.