Cloning of Rat Parkin cDNA and Distribution of Parkin in Rat Brain

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Abstract
The rat parkin cDNA sequence was characterized after screening a rat hypothalamus cDNA library with a 32P‐labeled probe containing the entire open reading frame of the human parkin cDNA. This sequence encompasses 1,576 bp and contains a single open reading frame that encodes a 465‐amino acid protein. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both the N‐terminal ubiquitin and the ring‐IBR (in between ring)‐ring finger domains appear to be highly conserved among rat, human, and mouse parkin. An affinity‐purified polyclonal antibody (ASP5p) was generated with a synthetic peptide corresponding to amino acids 295‐311 of the parkin sequence, which is identical in the three species. Western blotting revealed that ASP5p recognizes a single 52‐kDa band, which corresponds to the molecular mass of the parkin protein. Immunostaining with ASP5p showed that parkin is principally located in the cytoplasm of neurons that are widely distributed in the rat brain. Parkin‐immunoreactive neurons abound in structures that are specifically targeted in Parkinson's disease, e.g., subtantia nigra, but are also present in unaffected structures, e.g., cerebellum. Furthermore, parkin‐enriched glial cells can be detected in various nuclei of the rat brain. Thus, the role of parkin may be much more global than previously thought on the basis of genetic findings gathered in cases of early‐onset parkinsonism.