Porcine γ-synuclein: molecular cloning, expression analysis, chromosomal localization and functional expression
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The γ-synuclein protein is involved in breast carcinogenesis and has also been implicated in other forms of cancer and in ocular diseases. Furthermore, γ-synuclein is believed to have a role in certain neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. This work reports the cloning and characterization of the porcine (Sus scrofa) γ-synuclein cDNA (SNCG). The SNCG cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine SNCG cDNA codes for a protein of 126 amino acids which shows a high similarity to bovine (90%), human (87%) and mouse (83%) γ-synuclein. A genomic clone containing the entire porcine SNCG gene was isolated and its genomic organization determined. The gene is composed of five exons, the general structure being observed to be very similar to that of the human SNCG gene. Expression analysis by quantitative real-time RT-PCR revealed the presence of SNCG transcripts in all examined organs and tissues. Differential expression was observed, with very high levels of SNCG mRNA in fat tissue and high expression levels in spleen, cerebellum, frontal cortex and pituitary gland. Expression analysis also showed that porcine SNCG transcripts could be detected in different brain regions during early stages of embryo development. The porcine SNCG orthologue was mapped to chromosome 14q25–q29. The distribution of recombinant porcine γ-synuclein was studied in three different transfected cell lines and the protein was found to be predominantly localized in the cytoplasm.
KeywordsAnimal models Brain Embryogenesis γ-synuclein Pig SNP
Phosphate buffered saline
Single nucleotide polymorphism
We gratefully acknowledge Dr. Martine Yerle of INRA Toulouse, France for providing the pig-rodent hybrid panel. The authors wish to thank Connie Jakobsen Juhl and Helle Jensen for excellent technical assistance, Dr. Dorothy K. Madsen and Dr. Mark Henryon for critically reading of the manuscript and Dr. Rikke K.K. Vingborg for help with artwork. Our thanks are also due to Dr. Ole Højbjerg for assistance with confocal microscopy. This work was supported by a grant from the Danish Parkinson Association. The sequence of the porcine SNCG cDNA, encoding the g-synuclein protein, the entire genomic sequence of SNCG, and a short SNCG promoter sequence have been submitted to DDBJ/ EMBL/GenBank under the accessions numbers EF104640, EF104639 and EF486511, respectively.