Cloning, Bioinformatics And Tissue-Specific Expression Analysis Of Porcine JARID1C Gene

Jumonji, AT-rich interactive domain 1C (JARID1C) gene resides in the X chromosome, and it can escape X chromosome inactivation both in human and mouse. JARID1C protein belongs to the highly conserved ARID protein family, which involves in histone modification, chromatin remodeling and transcriptional regulation during cell growth, differentiation and development. Recent studies showed that, in human, this gene could play a vital role in normal brain development and function. The cDNA cloning and preliminary analysis of JARID1C gene not only provides primary information for better understanding the biological functions of JARID1C in pig, but also for the establishment of animal model to investigate human diseases in brain.In this study, using in silico approach combined with reverse transcription polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends (5' RACE), we cloned the full-length cDNA of JARID1C. Bioinformatics methods are adopted to predict the structure and function of JARID1C protein, and also to construct a phylogenetic tree to reveal the evolutionary relationship of various species. To check relative expression levels of JARID1C mRNA in various porcine tissues, the real-time PCR was performed.1 cDNA cloning and sequencing of porcine JARJD1C geneThe full-length cDNA sequence of human JARID1C gene served as an electronic probe, which was submitted to generate BLAST reciprocal best hits from dbEST and UniGene database, and about 20 EST sequences were retrieved. Using DNAstar-SeqMan program, these ESTs were assembled into two contigs. Based on the two contigs, the gene specific primers were designed by Oligo 6.0 for RT-PCR and RACE-PCR, which were carried out not only to check the validity of in silico approach, but also to obtain the full-length sequence of porcine JARID1C cDNA (5908bp). The sequence data were submitted to the GenBank databases under accession No. EF139241.2 Bioinformatics analysis of JARID1C proteinUsing bioinformatics network resources and relevant softwares, we predict that JARID1C cDNA has a 4548 bp open reading frame (ORF) flanked by a 522 bp 5' UTR and a 835 bp 3' UTR. The deduced amino acid sequence has 1516 residues, which contains no signal peptide, typical hydrophobic regions and transmembrane region, but is a nucleoprotein residing in nucleolus. A Conserved Domain Database alignment reveals that the protein contains several conserved domains, including a JmjN domain, an ARID domain, a JmjC domain, a C5HC2 zinc finger domain, a PLU-1 domain and a PHD zinc finger domain.3 Real-time PCR analysis of mRNA tissue-specific expression profilesAccording to the double standard curve method, real-time fluorescence quantitative PCR is performed to analyze the relative expression levels in various tissues. The results showed that JARID1C gene was expressed broadly in various tissues, but at very different levels. The strongest expression was observed in brain and gonad, whereas the lowest was seen in lung and skeletal muscle, suggesting that JARID1C protein play a role in porcine brain and gonad function.