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

Alan J. Nixon From the Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Nixon, Brower-Toland); and the Department of Orthopedics, Veterans Administration Medical Center, University of Washington, Seattle, WA 98108 (Sandell).

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Brent D. Brower-Toland From the Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Nixon, Brower-Toland); and the Department of Orthopedics, Veterans Administration Medical Center, University of Washington, Seattle, WA 98108 (Sandell).

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Linda J. Sandell From the Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Nixon, Brower-Toland); and the Department of Orthopedics, Veterans Administration Medical Center, University of Washington, Seattle, WA 98108 (Sandell).

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 PhD

Abstract

Objective

To isolate, clone, and determine primary nucleotide sequence of equine insulin-like growth factor I (IGF-I) and to examine IGF-I gene expression 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 comparison with other species. Total RNA from various tissues was probed with radiolabeled cDNA or complimentary RNA constructs by use of northern blotting, tube hybridization, or PCR procedures to determine IGF-I expression patterns.

Results

Nucleotide sequence of equine IGF-I was 90% homologous to that of cows, 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 splicing with retention of IGF-I exon 4 sequence, similar to rats and mice. Northern blot analysis revealed multiple IGF-I transcripts; predominant sizes were 1.6 and 4.5 kb. The IGF-I message was commonly detected in liver, kidney, 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 from those of other species, but the sequence coding the mature IGF-I peptide was more closely homologous. The larger IGF-IB form differed substantially in the carboxy-terminal. The biological action of the cleaved terminal was speculated to be autocrine feedback. Expression of IGF-I was apparent in many tissues, including cartilage, and was greater in immature horses. (Am J Vet Res 1999;60:1234–1241)

Abstract

Objective

To isolate, clone, and determine primary nucleotide sequence of equine insulin-like growth factor I (IGF-I) and to examine IGF-I gene expression 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 comparison with other species. Total RNA from various tissues was probed with radiolabeled cDNA or complimentary RNA constructs by use of northern blotting, tube hybridization, or PCR procedures to determine IGF-I expression patterns.

Results

Nucleotide sequence of equine IGF-I was 90% homologous to that of cows, 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 splicing with retention of IGF-I exon 4 sequence, similar to rats and mice. Northern blot analysis revealed multiple IGF-I transcripts; predominant sizes were 1.6 and 4.5 kb. The IGF-I message was commonly detected in liver, kidney, 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 from those of other species, but the sequence coding the mature IGF-I peptide was more closely homologous. The larger IGF-IB form differed substantially in the carboxy-terminal. The biological action of the cleaved terminal was speculated to be autocrine feedback. Expression of IGF-I was apparent in many tissues, including cartilage, and was greater in immature horses. (Am J Vet Res 1999;60:1234–1241)

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