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A heterologous system for exploring the localization of Arabidopsis tRNA nucleotidyltransferase

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A heterologous system for exploring the localization of Arabidopsis tRNA nucleotidyltransferase

Kanaan, Krystel (2007) A heterologous system for exploring the localization of Arabidopsis tRNA nucleotidyltransferase. Masters thesis, Concordia University.

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Abstract

The tRNA nucleotidyltransferase enzyme is crucial for normal tRNA maturation in eukaryotic cells. Sequence analysis revealed a possible nuclear localization signal (KDTKGKSIPVVNHIFKFSMKRK) in the Arabidopsis thaliana tRNA nucleotidyltransferase. We set out to examine the role of this sequence by expressing wild-type and variant Arabidopsis tRNA nucleotidyltransferase proteins in Saccharomyces cerevisiae. Viability assays implementing plasmid shuffling and complementation of a temperature-sensitive strain revealed that while the wild-type enzyme could replace the nucleocytosolic role of the yeast tRNA nucleotidyltransferase the variant enzymes could not. Subsequent enzyme activity assays revealed that enzyme activity was significantly reduced for the variant enzymes. Localization studies utilizing tRNA nucleotidyltransferase-green fluorescent protein fusion proteins showed a pattern of distribution inconsistent with the role of the modified region as a nuclear localization signal. In fact, the patterns observed seem to point to a possible stability-modulating function for our candidate region with a marked augmentation in fluorescence in the cytosol and nucleus in the variant proteins. Future experiments will be required to accurately define the role of this sequence in tRNA nucleotidyltransferase structure and function. Furthermore, additional studies are needed to define any nuclear localization signals that may be present in the Arabidopsis tRNA nucleotidyltransferase.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Thesis (Masters)
Authors:Kanaan, Krystel
Pagination:x, 73 leaves : ill. (some col.) ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Program:Biology
Date:2007
Thesis Supervisor(s):Joyce, Paul B. M
ID Code:975612
Deposited By: Concordia University Library
Deposited On:22 Jan 2013 16:11
Last Modified:18 Jan 2018 17:40
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