Genomic organization and expression of parkin in Drosophila melanogaster

Abstract

We report here the isolation, characterization on genomic structure and expression of the D. melanogaster homolog of human parkin. The 2,122 bp parkin gene sequence contains six exons that form a 1,449 bp transcript encoding a protein of 482 amino acids. 151 bp of 5' and 112 bp of 3' untranslated regions were identified by a combination of 5'-RACE/primer extension and X-RACE, respectively. The 5' UTR contains three transcription initiation sites. Neither a classical TATA nor a CAAT box was found in the putative promoter sequence. However, binding sites for AhR-Arnt, AP4, NF1 and GATA transcription factors were identified. Transient transfection analysis of the 5' UTR confirmed its promoter activity in HEK 293 cells and SH-SY5Y neuronal cells using a dual luciferase reporting system. The amino acid sequence of D. melanogaster Parkin exhibits 42%, 43% and 43% identity to that of human, mouse and rat, respectively, representing a 54 kDa protein band via western blot analysis. It shows a high degree of conservation in the Ubiquitin-like domain at the N-terminus (34%), the In-Between RING finger domains (IBR, 65-69%), and the RING finger domains at the C-terminus (56-57%). The expression pattern of D. melanogaster parkin varies during the developmental stages, with the highest expression in the adult stage as measured by competitive RT-PCR. From immunostainings of the embryo, D. melanogaster parkin was expressed slightly higher in the central nervous system (brain and nerve cord) during the late embryonic stage.