Characterization of histidine decarboxylase bearing an internal FLAG epitope in Drosophila melanogaste
College of Liberal Arts and Sciences
Medicine and Health Sciences
Histamine (HA) is synthesized by the enzyme histidine decarboxylase (HDC) and the analysis of mutants defective in HA synthesis has relied solely upon HA immunocytochemistry to identify HDC activity. Characterizing the expression of HDC has been hampered due to the inability to detect HDC immunocytochemically. HDC may also be post-translationally modified (i.e., removal of the N- and C-terminus (ref.1)), preventing the use of conventional N- and C-terminal epitope-labeling techniques. Previously, internal sites within the HDC gene structure were identified that can accept an insertion of a small epitope label, without disrupting the proteins function (ref. 2). Using this same approach, a FLAG epitope was placed in the same internal site of the Hdc genomic structure, resulting in the FLAG-Hdc gene. The FLAG-Hdc gene was placed into a transformation vector and transformed into Drosophila using standard injection techniques. Linkage of the FLAG-Hdc transgene was determined and selected transformant-bearing flies were crossed into a mutant HDC background lacking endogenous HDC activity (HdcJK910). These lines were analyzed for functional HDC protein by immunostaining larval brains with HA antibodies. A similar procedure was used to detect the epitope labeled FLAG-HDC protein using the M2 FLAG antibody. Results for the FLAG-HDC epitope immunodetection yielded a staining pattern very similar to the HA immunostaining, in a number of developmental stages. Co-localization experiments for both HA and FLAG-HDC were completed in the larval CNS using the HA and FLAG antibodies simultaneously, indicating that FLAG expression overlaps with HA localization. Future biochemical studies using this epitope tagged FLAG-Hdc transgene will be completed to further our understanding of HDC biochemistry and cell biology. (1) Fleming and Wang (2000) Molec. Cell Biol. 20: 4932 4947. (2) Fair et al. (2012) 53rd Drosophila Research Conference.
2014 Annual Drosophila Research Conference
San Diego, CA
Burg, Martin G.; Hage, Anthony; Mianecke, Maxwell; Trapp, Andra; and Burg, Deb, "Characterization of histidine decarboxylase bearing an internal FLAG epitope in Drosophila melanogaste" (2014). Faculty Scholarly Dissemination Grants. 966.