DNA and Protein Sequence Analysis of Neuronal Markers Neuronal Nuclei (Neun) and Doublecortin (Dcx) in the Northern Pacific Rattlesnake (Crotalus Oreganus) and Western Fence Lizard (Sceloporus Occidentalis).

Author

Brett M. Vassar, California Polytechnic State University, San Luis ObispoFollow

DOI: https://doi.org/10.15368/theses.2019.127
Available at: https://digitalcommons.calpoly.edu/theses/2527

Date of Award

6-2019

Degree Name

MS in Biological Sciences

Department/Program

Biological Sciences

College

College of Agriculture, Food, and Environmental Sciences

Advisor

Christy Strand

Advisor Department

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Advisor College

College of Agriculture, Food, and Environmental Sciences

Abstract

Neuronal Nuclei (NeuN) and Doublecortin (DCX) are neuron specific proteins that are used in histological studies of brain structure in a variety of vertebrate taxa.Antibodies against NeuN (anti-NeuN) bind to the Fox-3 protein, an RNA binding protein common in mature neurons. Anti-DCX labels a microtubule-associated protein expressed in actively dividing neural progenitor cells and migrating neurons. The DCX gene encodes a protein that is well conserved across mammalian, avian, and a few reptilian species, therefore anti-DCX staining has been used successfully across a range of vertebrate taxa. Successful neuronal staining using anti-NeuN has been demonstrated in mammals, birds, and the Testudines order (turtles). However, herpetologists who study neurobiology in squamates have had limited success with anti-NeuN and anti-DCX binding to their respective antigens. All commercially available anti-NeuN and anti-DCX antiserums were designed to mammalian antigens, and significant differences in tertiary structure divergence at the epitope where these antibodies bind may explain the failure of anti-NeuN and anti-DCX immunohistochemistry in many squamate species. This study aims to characterize evolutionary differences in gene and protein structure between two species of reptiles (Crotalus oreganus and Sceloporus occidentalis) and mammals. We sequenced the Fox-3 and DCX coding sequences using polymerase chain reaction (PCR) and Sanger sequencing, which allowed us to build phylogenetic trees comparing Fox-3 and DCX deduced protein structures. By identifying structural differences linked to evolutionary variation, new polyclonal antibodies specifically targeting Fox-3 and DCX in reptile brains can be developed to facilitate future investigations of neurogenesis and brain structure in squamate reptiles.