Benjamin Fair ACF Abstract FY12
Epitope labeling of histidine decarboxylase in Drosophila melanogaster
53rd Annual Drosophila Research Conference
Histidine decarboxylase (HDC) plays a critical role in the synthesis of histamine, a central and peripheral nervous system neurotransmitter used by invertebrates. Past attempts to create antisera that recognize HDC in vivo have not produced satisfactory results. While some HDC antisera have been made in other organisms, they appear not to be useful across species, including Drosophila melanogaster. As a result, little is known about the localization or biochemistry of HDC in the fly. It has been suggested that HDC undergoes a complex maturation process, undergoing cleavage at both the N- and C- termini of the protein. We report an approach that allows a functional HDC protein to be examined in vivo using internal epitope tagging. A genomic fragment that had been previously shown to contain a completely functional Hdc gene was modified by a PCR-mediated insertion of an epitope tag, 6x-HIS, into the protein coding region of the Hdc gene at specific sites. The location of these tags in the protein structure was selected to be in regions of the mature HDC protein which likely would not affect its function, based on comparisons of the structure of DDC from other species with the HDC protein sequence. Each Hdc transgene containing a 6X-HIS tagged Hdc gene was transformed into HdcJK910 mutant flies that normally have little to no histamine or HDC activity. Results indicate that while one of the epitope tags appears to disrupt Hdc function (indicated by a lack of histamine staining in the CNS), a 6X-HIS tag in a different location of the HDC protein structure appears to have no disruptive effect on Hdc function (indicated by normal histamine staining in the CNS). Assuming other epitopes can be used that may be easier to detect in tissue; this approach should enable further studies into the biochemistry and cell biology of HDC in vivo.
Benjamin Fair, Marc Vander Vliet, Stephanie Payne, and Martin Burg
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