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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.
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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)