Peer, Shawna Marie (2015-05). Characterization of GSTA3 Gene Products in Multiple Species and Demonstration of their Conservation in Divergent Species. Master's Thesis.
The presence and activity of glutathione S-transferase alpha 3 (GSTA3) in the testosterone biosynthesis pathway of humans and multiple mammalian species is a new concept. Some researchers have hypothesized that the gene encoding GSTA3 is conserved across many other species. This study undertakes characterization of the GSTA3 gene products in multiple diverse species in order to demonstrate its conservation. The species analyzed here include Canis lupus familiaris, Capra hircus, Monodelphis domestica, and Gallus gallus. Testis samples from each of these species were taken, RNA was isolated, reverse transcribed, and the GSTA3 cDNA was cloned and sequenced with polymerase chain reaction (PCR) technologies. cDNA sequences were then analyzed with the NCBI BLAST algorithm, ExPASy Translate tool, and Clustal Omega Multiple Sequence Alignment tool. These tools were also used to compare the cloned cDNAs to a reference sequence from Homo sapiens (GenBank accession number XM_000847.4) and an Equus caballus sequence published in the NCBI database (KC512384). GSTA3 cDNA sequences obtained for each species were submitted to NCBI GenBank. A set of multiple tissue samples was taken from both Equus caballus and Canis lupus familiaris. These tissue samples were analyzed for the expression of GSTA3 mRNA in each tissue after RNA isolation, reverse transcription and quantitative polymerase chain reaction (qRT-PCR). cDNA data analysis demonstrated GSTA3 gene conservation at the DNA level in all the species studied. Real time quantitative PCR data demonstrated significantly high concentrations of the GSTA3 mRNA in the testes and adrenal glands as compared to all other tissues for the equine multi-tissue analysis as expected. Dissimilarly, the canine multiple tissue analysis indicated that the concentration of GSTA3 mRNA was high in the liver and small intestine as well as the testes and adrenal glands. Other researchers have reported high concentrations of GSTA3 mRNA in the liver and small intestine previously.