Molecular cloning, expression and localization of a putative Drosophila melanogaster farnesoic acid o-methyltransferase.

摘要

Farnesoic acid o-methyltransferase (DmFAMeT) is an enzyme that converts farnesoic acid to methyl farnesoate in the final steps of juvenile hormone biosynthesis in Drosophila melanogaster . DmFAMeT was cloned and encoded for an open reading frame of 291 amino acids with a molecular weight of 31.4 kDa. The comparison of DmFAMeT with sequences from putative crustacean FAMeTs indicated regions of high similarity. The recombinant DmFAMeT (rDmFAMeT) was used for the production of anti-DmFAMeT serum. The rDmFAMeT lacked FAMeT activity. Addition of rDmFAMeT with a corpora allata from Diploptera punctata did not appear to increase FAMeT activity levels above that observed by the CA extract control. This may suggest that post-translational modification may be required for activation. RT-PCR, demonstrated that the DmFAMeT gene expressed one transcript in larval, pupal and adult stages. DmFAMeT expression was monitored by use of a DmFAMeT promoter fusion with the lacZ reporter gene that was cloned in a P-element vector which was used to make germline transformants. Two regions of DmFAMeT promoter were cloned. The first construct contained sequences from -1171 to -97 and the second -1171 to +548 which also included the first intron. DmFAMeT germline transformants revealed no discernable difference on lacZ expression compared to wild type controls. (Abstract shortened by UMI.)