The Histidine decarboxylase (Hdc) gene of Drosophila melanogaster is responsible for the synthesis of histamine in both the brain and peripheral tissues. Recently, two mRNA isoforms of the Hdc gene were identified, which differ at either end of the mRNA molecule. The goal of this project is to determine whether Hdc mRNA isoform expression is tissue-specific, potentially reflecting the difference in histamine distribution in the adult fly. For this project, two regions of the Hdc gene are being used for anti-sense riboprobe synthesis: one probe should bind to all Hdc mRNAs, and the other probe should bind specifically to a unique mRNA isoform. Using PCR, we amplified a 230 base pair fragment from the Hdc gene that is unique to only one mRNA isoform and used TA cloning to insert this fragment into the pGEM-T Easy vector. Then, the DNA from the transformant cells was isolated, sequenced, and linearized using specific restriction endonucleases. The linearized DNA template was subsequently used with the Invitrogen FISH Tag RNA kit to synthesize an amine-modified RNA that should bind to all Hdc mRNAs. Then, the amine-modified RNA was labeled with the fluorescent dye Alexa fluor 488. We are currently synthesizing the 230 bp isoform-specific riboprobe with Alexa fluor 594, so that double labeling experiments with both mRNA probes may be accomplished. To complete this project, both RNA probes will be hybridized to adult Drosophila tissue sections and the resultant signals can be detected using fluorescence microscopy. Results are expected to demonstrate that one mRNA isoform is solely expressed by histaminergic neurons in the CNS, while the other is only expressed in the peripheral tissues, such as photoreceptors. In the future, we plan to expand this project by using tissue sections of flies at different developmental stages.
Faculty Mentor: Martin Burg, Biomedical Sciences