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Date of Award

4-24-2021

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Arts

Department

Biology

First Advisor

Dr. Angie Hilliker

Abstract

Ded1 is an essential DEAD-box protein in yeast involved in translation. Specifically, it is necessary for translation initiation. Ded1 can be methylated at four arginine residues, which has the potential to affect its functionality. It is important to identify when Ded1 is methylated in vivo and what conditions may cause a change in the level of methylation in Ded1. An in vivo assay was developed using an anti-monomethyl arginine antibody to selectively bind Ded1 from samples. When using this in vivo methylation assay to determine methylation of Ded1 in various growing phases, the anti-monomethyl probe was unable to differentiate between methylated and unmethylated Ded1 in vivo. A FLAG-tag immunoprecipitation was used to confirm that the antimonomethyl antibody is selecting Ded1 and not a monomethylated protein of similar size. The monomethyl arginine was able to differentiate between recombinant methylated and unmethylated Ded1 protein purified from E. coli and was able to differentiate between wildtype and an unmethylated mutant form of Ded1 from yeast. Additionally, more cellular stress conditions will be tested using the in vivo methylation assay.

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