Porcine Parkin: molecular cloning of PARK2 cDNA, expression analysis, and identification of a splicing variant

Abstract

Parkin, encoded by the PARK2 gene, is an E3 ligase which functions as an integral component of the cytoplasmic ubiquitin/proteasomal protein degradation pathway. Mutations in the PARK2 gene, resulting in the loss of parkin function, leads to autosomal recessive juvenile Parkinsonism (AR-JP). This work reports the cloning and characterization of the porcine (Sus scrofa) PARK2 cDNA (SsPARK2) and splicing variants hereof. The PARK2 cDNA was amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine PARK2 cDNA codes for a protein of 461 amino acids which shows a high similarity to orangutan (91%), human (86%), and to rat (82%) parkin. A splicing variant of the porcine PARK2 with a complete deletion of exon 9 was also identified. Expression analysis by quantitative real-time RT-PCR revealed presence of PARK2 transcript in all examined organs and tissues. Differential expression was observed, with very high levels of PARK2 mRNA in cerebellum, heart, and kidney. In addition, expression analysis showed that porcine PARK2 transcripts could be detected early in embryo development in different brain regions. The porcine PARK2 orthologue was mapped to chromosome 1p24-25. Single nucleotide polymorphism (SNP) analysis revealed seven SNPs in the porcine PARK2 gene, one missense and one silent mutation in exon 7 and five SNPs in intron 7.