Date Approved

5-2020

Graduate Degree Type

Thesis

Degree Name

Biomedical Sciences (M.H.S.)

Degree Program

Biomedical Sciences

First Advisor

Dr. Merritt DeLano-Taylor

Second Advisor

Dr. John Capodilupo

Third Advisor

Dr. Derek Thomas

Fourth Advisor

Dr. Daniel Bergman

Academic Year

2019/2020

Abstract

One of the leading regulators of neuronal cell differentiation in the CNS is the family of basic helix–loop–helix (bHLH) transcription factors. One of these proteins, Nato3, is associated with the formation of dopaminergic neurons. Transcription factors can be regulated by kinase activity, and in order to detect the associated change of phosphorylation of Nato3, we have generated Nato3 with specific epitope tags that allow for the detection and isolation of the Nato3 protein. Through subcloning techniques and a successful transformation of the Nato3 gene with the sequences of the 3X-Flag epitope and the Myc epitope into the pcDNA3.0 vector, we were able to begin testing the phosphorylation status of Nato3. In particular, these epitopes attached to Nato3 allowed us to better detect the phosphorylation status and migration patterns in SDS-PAGE gels. We were able to see significant mobility shifts on the gels consistent with a phosphorylation event. This type of phosphorylation of the Nato3 protein will allow us to continue forward to determine the specific residue within Nato3 that is phosphorylated. Understanding the post-translational modification of Nato3 may help lead to better understanding of dopamine neurogenesis.

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