A porcine ortholog of this gene has been described around the mRNA level (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_214148″,”term_id”:”1205916046″,”term_text”:”NM_214148″NM_214148) and first functional analyses have been performed (Loewen et al. that appear to be species-specific have been reported for the human (Kamada et al. 2004) and the equine (Anton et al. 2005). Fourth, the murine mCLCA6 protein is expressed in different cell types and in different subcellular structures than its direct human ortholog, hCLCA4 (Bothe et al. 2008). Moreover, the first and only porcine CLCA protein identified to date, pCLCA1 (Gaspar et al. 2000), displayed different functions and electrophysiological properties when compared with its human and murine orthologs (Loewen et al. 2002b). Thus, a detailed understanding of the porcine pCLCA1 and possible pig-specific variations in the gene family appears critical before their role as modulators of the CF phenotype can be studied and interpreted in the promising new pig models. The aim of this study was to characterize the genomic organization of the porcine gene, its protein expression pattern, and its posttranslational protein modification and trafficking. The results are compared with the corresponding human and murine orthologs to disclose differences that could be relevant for the interpretation of porcine CF models. Materials and Methods Characterization ABT-239 of the Genomic Structure and Other Porcine Genes The organization of genes in mammals was evaluated by the GenBank DNA database (http://www.ncbi.nlm.nih.gov/). Porcine bacterial artificial chromosomes (BACs) notionally corresponding to the human locus were identified by comparison of the human genome with pig BAC end sequences. Subsequently, the candidate BACs were located on the porcine genome by the pig fingerprint contig map (www.ensembl.org). Four BAC clones covering the complete porcine locus, including the flanking genes and (CH242-32G22, CH242-148M17, CH242-252F2, and CH242-483E7 with GenBank accession Rabbit polyclonal to ACMSD numbers “type”:”entrez-nucleotide”,”attrs”:”text”:”CU695058″,”term_id”:”260161802″,”term_text”:”CU695058″CU695058, “type”:”entrez-nucleotide”,”attrs”:”text”:”CU694822″,”term_id”:”260207454″,”term_text”:”CU694822″CU694822, “type”:”entrez-nucleotide”,”attrs”:”text”:”CU695038″,”term_id”:”260161803″,”term_text”:”CU695038″CU695038, and “type”:”entrez-nucleotide”,”attrs”:”text”:”CU469041″,”term_id”:”260161805″,”term_text”:”CU469041″CU469041, respectively), were obtained from CHORI BACPAC resources center (http://bacpac.chori.org/) and sequenced by the Wellcome Trust Sanger Institute (Hinxton, UK). Genes were roughly localized around the contig sequence by comparison of designated mRNA sequences from pig, human, cow, horse, mouse, and doggie to the porcine BACs by BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Predicted porcine mRNA sequences were derived from the alignment of porcine ABT-239 BACs and mRNA sequences from other species using BioEdit and taking into account the exon-intron structure in the different species as well as putative splicing sites in the BACs (Hall 1999). The corresponding protein sequences were deduced from the predicted mRNA sequences by in silico translation. Phylogenetic trees of CLCA amino acid sequences from different species were generated by the PHYLIP software package (http://evolution.genetics.washington.edu/phylip.html), and nomenclature of the porcine genes was assigned by their correlation to the major branches of the trees. Animals and Tissue Processing Tissues from five male pigs (6 weeks old, EUROC Pietrain), two female pigs (2 and ABT-239 3 months old, mixed breed), and one male pig (7 months old, mixed breed) that had been euthanized for other reasons were included in this study. The following tissues were immersion fixed in 4% neutral-buffered formaldehyde or shock-frozen in liquid nitrogen after brief immersion in 2-methylbutane: nasal cavity, larynx, trachea, lung (three different locations: cranial left lobe, left main lobe, accessory lobe), tracheal bronchus, left principal bronchus, esophagus, stomach (glandular and non-glandular parts), duodenum, jejunum, ileum, cecum, colon, rectum, parotid salivary gland, pancreas, liver, gall bladder, kidney, urinary bladder, mandibular lymph node, spleen, heart, aorta, brain (cortex, cerebellum, medulla), eyes, skin (perineum, rooting disc, prepuce), testicle, epidymides, spermatic cord, uterus, and ovary. Cloning and Sequencing of pCLCA1 cDNA Total RNA was extracted from porcine rectum using the Trizol method (Invitrogen; Karlsruhe, Germany) and purified using the RNeasy Mini Kit (Qiagen; Hilden, Germany).