Date Approved

12-2014

Graduate Degree Type

Thesis

Degree Name

Cell and Molecular Biology (M.S.)

Degree Program

Cell and Molecular Biology

First Advisor

Dr. Martin Burg

Second Advisor

Dr. Matthew Christians

Third Advisor

Dr. Merritt Taylor

Fourth Advisor

Dr. Georgette Sass

Abstract

Histamine is a neurotransmitter in arthropods and is responsible for synaptic transmission in vision, mechanosensation, temperature sensing and sleep cycle in Drosophila. Histamine is synthesized by the enzyme histidine decarboxylase (HDC). While histamine is detectable within tissues using current immunofluorescent labeling techniques, immunological approaches have not been successful for HDC itself, with both direct antibodies and terminal epitope tags determined to be ineffective. In order to avoid loss of the epitope tag through putative N-­‐ and C-­‐terminal proteolytic cleavage, known to occur for HDC in other organisms, an internal epitope tag that does not disrupt enzyme function was utilized. A 6xHIS internal epitope tag had been previously employed and though it was not successful as a method to detect the HDC protein, it did demonstrate that an internal SacI restriction site could be used without disrupting the protein’s function and a FLAG epitope tag was employed instead. We found that an internally located HDC-­‐FLAG tag is an effective method for determining the location of HDC in a variety of tissues. In the brain of Drosophila, HDC is found to co-­‐localize with histamine in histaminergic cells and structures. In larvae, HDC was found to localize to the same 10 pairs of cells in the ventral nerve cord found to be histaminergic. In adults, HDC was detected in central brain neuropil region where synaptic terminals are located, cells in the central brain, and photoreceptors as well. Photoreceptor localization was not as strong as expected, displaying a punctate staining pattern in the photoreceptors. Embryo analysis showed very restricted HDC localization, except for a novel pattern on their lateral edges, postulated to be the chordotonal organs. While the digestive tract displayed immunofluorescence in adult tissue sections, whole mount gut immunofluorescence experiments stained negative for FLAG. In summary, the internal HDC-­‐FLAG epitope tag has been shown to be an effective, useful tool for HDC detection in tissue and has the capacity for enabling new discoveries in histamine biology in Drosophila.

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