Cloning of rat parkin cDNA and distribution of parkin in rat brain

The rat parkin cDNA sequence was characterized after screening a rat hypoth alamus cDNA library with a P-32- labeled probe containing the entire open r eading frame of the human parkin cDNA. This sequence encompasses 1,576 bp a nd contains a single open reading frame that encodes a 465-amino acid prote in. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both t he N-terminal ubiquitin and the ring-IBR (in between ring)-ring finger doma ins appear to be highly conserved among rat, human, and mouse parkin. An af finity-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 rec ognizes a single 52-kDa band, which corresponds to the molecular mass of th e parkin protein. Immunostaining with ASP5p showed that parkin is principal ly located in the cytoplasm of neurons that are widely distributed in the r at brain. Parkin-immunoreactive neurons abound in structures that are speci fically targeted in Parkinson's disease, e.g., subtantia nigra, but are als o present in unaffected structures, e.g., cerebellum. Furthermore, parkin-e nriched glial cells can be detected in various nuclei of the rat brain. Thu s, the role of parkin may be much more global than previously thought on th e basis of genetic findings gathered in cases of early-onset parkinsonism.