2015 IPFW Student Research and Creative Endeavor Symposium
 

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Faculty Sponsor

Dr. George Mourad

Department/Program

Department of Biology

University Affiliation

Indiana University – Purdue University Fort Wayne

Abstract

Nucleobases are important organic molecules, and are involved in DNA biochemistry, sugar metabolism, and serve as energy molecules.Catabolism and biosynthesis of these molecules takes place in different compartments of a cell, and they must be transported between cells.Thus, an intricate system of membrane transporters is used.Several families of nucleobase transporters exist, and one such family is the Nucleobase Cation Symporter 1 (NCS1) family.Bioinformatics revealed a single gene encoding for a putative NCS1 in Nicotiana sylvestris (NsNCS1), a dicot angiosperm commonly known as tobacco.In this study, the substrate profile of this putative transporter was determined.The coding region of the gene was cloned into a yeast expression vector and transformed into Saccharomyces cerevisiae strains deficient in their native NCS1 transporters.Radiolabeled nucleobase uptake experiments were used to determine the transport profile.The binding profile was determined through heterologous competition using a cold competitor.Homologous competition experiments were used to determine kinetic properties of NsNCS1.In addition, the function of NsNCS1 was confirmed by complementing the NCS1 function in a homozygous Arabidopsis thaliana AtNCS1 knock-out mutant constructed in our lab.These experiments revealed that NsNCS1 transports adenine, guanine, uracil, hypoxanthine, and cytosine, and binds 5-fluorocytosine, uric acid, and xanthine.This profile resembles the transport and binding profiles of other Viridiplantae NCS1 proteins studied in our lab.It highlights the core functionality of Viridiplantae NCS1 proteins, and given the taxonomic position of Nicotiana sylvestris, sheds light on the evolution of NCS1 function.

Disciplines

Biology | Life Sciences

Functional characterization of the Nucleobase Cation Symporter 1 (NCS1) of Nicotiana Sylvestris

Included in

Biology Commons

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