Date of Award

8-21-2017

Degree Type

Thesis

Degree Name

Biomedical Sciences (M.H.S.)

Department

Biomedical Sciences

First Advisor

Martin Burg

Second Advisor

Georgette Sass

Third Advisor

Osman Patel

Academic Year

2016/2017

Abstract

Histamine is a biogenic amine that functions as a neurotransmitter in a number of vertebrate and invertebrate systems and is synthesized from its precursor histidine by the enzyme histidine decarboxylase (HDC). In Drosophila, histamine has been shown to have function in photoreceptors, mechanoreceptor cells, as well as centrally located neurons. Mutations of the Hdc gene, such as HdcJK910, exhibit defects in histamine synthesis and display altered behaviors such as blindness, inability to groom, impaired thermal tolerance, and altered sleep rhythms. However, all Hdc mutants obtained thus far demonstrate some transcriptional activity.

In order to remove Hdc expression completely, part of the Hdc gene was removed via Minos transposon-excision mutagenesis using the Mi{ET1}HdcMB07212 fly, which bears a Minos transposon within the Hdc gene (a hypo-morphic allele). Minos excision mutagenesis of Hdc was achieved by mating flies from the HdcMB07212 strain with another fly carrying the Minos-specific transposase gene – to induce imprecise excision in the progeny's genome to cause a deletion. The Mi{ET1} transposon also contains a gene encoding the green fluorescent protein (GFP) under the control of an eye-specific promoter, the loss of which can be used to visually identify a potential Minos excision. Once loss of GFP (GFP-) individuals were identified, breeding lines were established and flies from each line examined using histamine immunostaining to determine the presence or absence of histamine in the ventral nerve cord of larvae. Progeny obtained fell into the following categories: (1) flies with wild-type levels of histamine, indicating rescue of Hdc expression due to a precise Minos excision from Hdc; (2) flies with trace levels of histamine, indicating an excision event that disrupted GFP expression from the Mi{ET1} transposon but did not rescue Hdc expression; (3) flies having no histamine, 4 indicating an imprecise excision with an associated loss of expression from the Hdc gene. Molecular lesions associated with each class of flies were characterized using a PCR approach. Results indicate that of the 98 GFP- strains examined thus far each fell into one of the 3 expected categories, with 29 exhibiting elimination of Hdc expression.

Available for download on Friday, August 24, 2018

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