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Sequence Evolution of IGF-1 in Snakes Courtney 1 Hewitt , Trinket snake (Elaphe helena) 2 Sparkman , 2 Serb 2 Bronikowski Amanda Jeanne & Anne 1Newton High School, Newton IA 2EEOB Department, Iowa State University, Ames, IA Cat-eyed snake (Madagascarophis colubrina) Western terrestrial garter (Thamnophis elegans) Checkered garter (Thamnophis marcianus) Introduction Insulin-like growth factor-1 (IGF-1) is a highly conserved peptide hormone that stimulates growth and reproduction, and negatively impacts lifespan. 1 It has been studied widely in mammals, chickens, and fish, but few studies on IGF-1 have been conducted with reptiles. In our lab, when we attempted to validate a hormone assay using a human anti-IGF-1 antibody in a variety of species of snake, some species would bind predictably to the antibody whereas others would not.2 Crotaliae VIPERIDAE Viperinae Agkistrodon contortrix Results Causus defilippi Elaphe guttata Elaphe helena Spalerosophis diadema Lamprophis fuliginosus Thamnophis elegans Thamnophis marcianus Colubrinae COLUBROIDEA Natricinae Pseudoxyrhophiinae We found twenty-six different haplotypes of IGF-1. Nineteen of these were found only in a single individual, while seven different haplotypes appeared repeatedly within and among species (Fig 2). Individuals had anywhere from 1-5 haplotypes of IGF-1. Many of the amino acid changes in these haplotypes were in regions that are highly conserved in other species, suggesting the possibility that they may alter the protein shape. Madagascarophis colubrina We hypothesized that there has been evolution in the IGF-1 gene sequence in snake lineages that has resulted in amino acid changes that may have altered the shape of the IGF-1 protein. Fig 1. Phylogeny of snakes sampled 3 Conclusion Methods We sequenced IGF-1 from liver mRNA in 1-3 individuals of nine species distributed across the snake phylogeny (Fig1). We designed nested primers for IGF-1 based on conserved regions from human and chicken IGF-1 sequences on GenBank. We extracted mRNA from liver and synthesized cDNA. Followng PCR we performed a bacterial transformation by inserting our gene into a vector and raising it up in E.coli. After choosing bacterial colonies containing our insert, we performed a sequencing reaction and sent the samples to the DNA sequencing facility. We analyzed the nucleotide sequences of the cDNA to assess non synonymous nucleotide changes. 21 26 34 39 40 43 52 # sp # ind R S S S T I P 6 10 R S S S T I L 5 8 R S N S T I L 3 6 R S S S T T L 3 3 G S S S T I L 3 3 R G S S T I L 2 2 R S S Y S I L 2 2 Fig 2. Haplotypes of IGF-1 found in multiple species and individuals. Night adder (Causus defilippi) Copperhead (Agkistrodon contortrix) African house snake (Lamprophis fuliginosus) We found abundant variety in the IGF-1 amino acid sequence within and among individuals and species (Fig 2). This suggests that there has been remarkable diversification in IGF-1 in snakes that has not been reported in any other group yet examined. This research will lead to a more in depth comparative studies to assess IGF-1 evolution, and how that evolution may have resulted in changes in protein shape. Literature Cited 1. Reviewed in Bartke, 2005. Endocrinology 146:3718-3723 2. Sparkman et al., unpublished data 3. From Lawsen et al., 2005. Mol. Phyl & Evol. 37:581-601 Egyptian diadem (Spalerosophis diadema) Corn snake (Elaphe guttata)