A study of parapinopsin in amphibians

Abstract

Parapinopsin (PNP) is a novel pigment protein present in pineal gland of the brain. PNP protein is similar in function to the human visual pigment protein rhodopsin, found in the eye. The aim of project is to sequence and characterize PNP in different frogs species to understand more about PNP as little is known about its structure and function.
Tadpole cDNA from different frogs species (X.laevis, Rana temporaria and Rana tigrina) were used during the project. PCR primers were designed against Xenopus tropicalis PNP cDNA the only reported sequence at NCBI. PNP PCR products were cloned into pGEM vectors and sequenced. The cDNA sequencing results of X. laevis showed 100% homology but R. temporaria and R. tigrina showed ~99.9% similarity with X. tropicalis. The 0.1% dissimilarity is due to a point mutation from G to A at the 2nd position in the 156th codon. Genomic DNA sequence study results revealed a heterozygosity in R. temporaria and a point mutation in R. tigrina PNP. This point mutation is very important as it creates a stop codon possibly resulting in protein truncation. These findings were further confirmed by SSP-PCR (sequence specific primers- PCR) results. Western blotting was used to determine more about the size of the protein produced. Two peptides regions were selected at N and C termini. Western Blotting results support the cDNA study results with a 37 kDa protein band of PNP in the X. laevis protein sample only. Bioinformatics softwares were used to study evolutionary genetics and to predict the 2D structural elements and a 3D structural model for PNP from X. laevis. Further studies were conducted on X. laevis melanophores to look for PNP expression. Northern Blotting results indicated the absence of PNP in melanophores. For protein expression studies the entire coding sequence of PNP gene was chemically synthesized and subsequently cloned into the vector pASKGPCR for expression in E. coli. The aim of this study was to overexpress and purify the protein for functional studies as well as structural studies using X-ray crystallography and high field NMR.

Date of Award	2014
Original language	English
Awarding Institution	King's College London
Supervisor	Peter Eagles (Supervisor) & Mark Sanderson (Supervisor)