Exploration of HRP-biosynthetic pathway through two mutations in Drosophila melanogaster
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Anti-HRP staining of two D. melanogaster mutants generated from an ethyl methane sulfonate (EMS) mutagenesis, sugar-free frosting (sff) and MS16-2, revealed a decrease in embryonic HRP-epitope expression. MS16-2 embryos and w1118 embryos, serving as a control, were prepared into protein powder and subsequently digested with peptide N-glycosidase (PNGase) F and A. The released glycans were analyzed with nanospray ionization-linear ion trap mass spectrometry and collision-induced dissociation spectrometry. The N-glycan profile of both preparations showed similar glycan species and prevalence; both profiles contained predominantly high mannose and pauci-mannose species. Examination of the prevalence of HRP epitopes between the two samples reveals that there is a decrease in HRP-epitopes in MS16-2. Using [13C] methyl iodide and [12C] methyl iodide to differentially label glycans during permethylation, the relative abundance of N-linked glycans can be quantified and compared between w1118 and MS16-2 embryos. The differential labeling analysis also showed a decrease in HRP-epitope expression in MS16-2, confirming the results of the anti-HRP staining. Brains dissected from third-instar sff and w1118 larvae were prepared into protein powder and subsequently analyzed for N-glycans. The N-glycan profile of the sff larval brains showed a decrease in HRP-epitopes, suggesting that the defect in fucosylation continues into the larval stage. Heads harvested from w1118, w-; sff, OreR, and w+; sff adult males were also analyzed for N-glycans. N-glycan analysis of the heads also showed a decrease in HRP-epitope expression in sff adult heads, indicative of sustained defective fucosylation activity through the adult stage. Geotaxis testing was performed on sff and MS16-2 adult males to examine the effects of the mutation. Wild type flies that go through geotaxis testing escape out of their vial within 15 seconds. Flies that were sff, however, displayed a less dynamic behavior, with the majority of flies staying in the vial even after given 120 seconds to escape. MS16-2 flies showed a similar behavior to sff, but not as severe as sff. The results of the geotaxis testing served as a basis for developing a geotaxis mutant screen. Mutants generated from an EMS mutagenesis are screened through an escape test. The flies that fail to escape are further tested in order to isolate mutations that affect geotaxis behavior, with hopes of discovering mutations that affect different aspects of the HRP biosynthetic pathway and ultimately elucidating more about the mechanisms of the pathway.