Genomic organization and expression of parkin in Drosophila melanogaster

다국어 초록 (Multilingual 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 3'-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.