Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an imp ortant arthropod immune protein. In mosquitoes, prophenoloxidase has been s hown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vecto r, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5' flanki ng sequence (-855 bp) of the prophenoloxidase 1 gene, was determined. The g ene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA, In the 5' flanking sequence, we found several putative regula tory motifs, two of which were identified as ecdysteroid regulatory element s. Electrophoretic mobility gel-shift assays and supershift assays demonstr ated that the Aedes aegypti ecdysone receptor/ultraspiracle nuclear recepto r complex, and, seemingly, the endogenous Anopheles gambiae nuclear recepto r complex, was able to bind one of the ecdysteroid response elements. Furth ermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcr iption of the prophenoloxidase 1 gene in an A. gambiae cell line.