Abstract:
The aim of this Master’s thesis was to evaluate the hypothesis that the Saccharomyces cerevisiae homologs for Eukaryotic Initiation Factor 4A, TIF1 and TIF2, can be individually regulated. This may be an expectation if the retention of the TIF1 and TIF2 duplicates arises from the requirement to respond to a wide variety of cellular needs in S. cerevisiae. These paralogs show an almost identical sequence in their coding region (barring six synonymous changes in the gene sequence) but are substantially different in their 5’ and 3’ untranslated regions that are the probable sites of regulatory functions. To identify differences in use of the TIF1 and TIF2 paralogs, a dual fluorescent reporter strain expressing plasmid borne TIF1-RFP, incorporating the endogenous TIF1 3’ and 5’ untranslated regions, and chromosomally integrated TIF2-GFP was created in order to probe for any differential regulation between TIF1 and TIF2. To create the fluorescent reporters it was necessary to learn and execute sophisticated molecular biology and molecular genetics which are described in this thesis. The generated fluorescence reporter strains were shown to be stable over multiple generations and subjected to high throughput and high content automated confocal microscopy. The commercially available LOPAC1280 Library of Pharmacologically-Active Compounds was used to probe for differential regulation where a “hit” was defined as a significant change in the expression of at least one of TIF1-RFP or TIF2-GFP four hours after application. For TIF2p-GFP, 2 compounds out of the 1280 library showed evidence of regulation under stringent thresholding criteria. For TIF1-RFP, 43 compounds were identified as regulators. There was no overlap of compounds. This screen provides prima-facie evidence that the TIF1 and TIF2 are differentially regulated, as assessed by the criteria of the experimental system described in this thesis.
