Degree Name

BA

Department

Microbiology and Molecular Biology

College

Life Sciences

Defense Date

2020-03-04

Publication Date

2020-03-19

First Faculty Advisor

Dr. Jonathon Hill

First Faculty Reader

Dr. Steven Johnson

Honors Coordinator

Dr. R. Paul Evans

Keywords

cis-regulatory, characterization techniques, upstream promoters

Abstract

This thesis examines a novel technique for characterizing promoters using a zebrafish model. The proximal upstream cis-regulatory elements, also known as promoters or promoter regions, are essential for the precise regulation and timing of gene expression. Often the characterization of these regions relies on imprecise methods involving large deletions or bioinformatic predictions rather than experimental data. However, high-throughput sequencing technology could potentially allow large libraries containing hundreds of thousands of variants of a single promoter to be simultaneously analyzed. We have been working to develop a novel method for promoter characterization that takes advantage of this technology. We tested this method by producing a variant library of the zebrafish cardiac myosin light chain-2 gene (cmlc2) promoter, then used those variants to drive expression of a degenerate barcode in the 5’ UTR of gfp. We found that tracking the relative expression levels of the barcodes allowed us to successfully characterize the cmlc2 promoter in line with a previously published analysis using less precise techniques. Our method also led to discoveries of additional sites of gene regulation within the cmlc2 promoter. We next designed improvements to the process to improve workflow and adaptability to other promoters. We tested these improvements with the zebrafish ventricular myosin heavy chain (vmhc) promoter.

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