Characterization of Farnesoic Acid O-Methyltransferase (FAMeT) and Juvenile Hormone Acid Methyltransferase (JHAMT) in relation to Drosophila melanogaster Juvenile Hormone Biosynthesis
Burtenshaw, Sally M.
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Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. The role of two key enzymes in the biosynthetic pathway of JH were examined: Farnesoic Acid O-Methyltransferase (FAMeT) and Juvenile Hormone Acid Methyltransferase (JHAMT). In crustaceans, FAMeT has been found to methylate farnesoic acid (FA), producing methyl farnesoate (MF) prior to epoxidation at the penultimate stage of JH biosynthesis. JHAMT was discovered more recently in the silkworm Bombyx mori and converts epoxidated FA (JHacids) to active JH through methylation using S-adenosyl-L-methionine (SAM). The aim of the proposed research is to examine the influence of a) decreasing the amount of FAMeT produced using an enhancer trapping P-element and b) increasing the levels of JHAMT and FAMeT in specific tissues using GAL4 overexpression techniques. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of D. melanogaster ring glands. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on the sesquiterpenoid biosynthesis. Drome-AST appears to have a select effect on JHB3 biosynthesis and not MF or JHIII. Analysis of JHB3 release from larval and adult flies ubiquitously overexpressing JHAMT showed a significant increase when compared to wildtype (p<0.01 and p<0.0001 respectively). No significant difference was seen in JHB3 release in flies ubiquitously overexpressing FAMeT. A significant increase in hatching success was seen in flies overexpressing FAMeT in the larval ring gland and oocytes (p<0.05) whereas no significant decrease was seen in JHAMT-overexpressing flies during development. A significant extension of lifespan was also seen when FAMeT was overexpressed in the border and follicle cells of the oocyte (p<0.0001). The direct role of JHAMT in JHB3 synthesis has been demonstrated. The involvement of FAMeT and JHAMT in development and longevity may require other interacting proteins to elicit an effect, which is a limiting factor in overexpression experiments of the two enzymes. Additionally, this is the first example of AST action within D. melanogaster.