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Widely used antipig CD4 monoclonal antibodies (mAbs) fail to recognize CD4 alleles characteristic of miniature pig lines such as the National Institutes of Health (NIH) miniature pigs and microminipigs. We surveyed polymorphisms in the coding sequence of the porcine CD4 gene among Western and Oriental pig breeds and Japanese wild boars and investigated their distribution. Of the 13 alleles that we identified among the 47 animals, 2 in group I and 3 in group II were found exclusively in Western breed pigs. Group IV alleles, which included mAb-nonbinding alleles, were found frequently in Oriental breed pigs, suggesting that the mAb-nonbinding allele arose from the gene pool of Oriental pigs. Group IV alleles were also found in Duroc and Large White pigs, suggesting genetic inflow from Oriental pig breeds into Western breeds. Comparison of the CD4 sequences of species in Cetartiodactyla suggested that the group IV alleles in Sus scrofa occurred before the divergence of this species from the other artiodactyls. The different antibody specificities of the various CD4 alleles may facilitate the discrimination of T-cell populations in transplantation studies using miniature pigs. The significance of the preservation of CD4 polymorphisms to immune function in pigs warrants further investigation.

Nucleotide-binding oligomerization domain 1 (NOD1) is a cytosolic pattern recognition receptor that recognizes γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), a component of bacterial peptidoglycan. NOD1 is thought to be involved in the immune homeostasis mediated by intestinal microbiota as well as the host defense against infection. In this study, we identified 12 synonymous and nine nonsynonymous single nucleotide polymorphisms (SNPs) in the coding sequence of porcine NOD1 within major commercial breeds in the swine industry. Among the nonsynonymous SNPs, two amino-acid alterations located in the leucine-rich repeats region, glycine to glutamic acid at position 641 (G641E) and aspartic acid to asparagine at position 918 (D918N), impaired iE-DAP-induced activation of nuclear factor-κB. These alleles showed the recessive mode of inheritance and therefore are likely to be maintained in pig populations at high frequencies. These results suggest the possibility for improvement in disease resistance by eliminating the G641E and D918N alleles of NOD1 from commercial pig populations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Along with the draft sequencing of the pig genome, which has been completed by an international consortium, collection of the nucleotide sequences of genes expressed in various tissues and determination of entire cDNA sequences are necessary for investigations of gene function. The sequences of expressed genes are also useful for genome annotation, which is important for isolating the genes responsible for particular traits., Results: We performed a large-scale expressed sequence tag (EST) analysis in pigs by using 32 full-length-enriched cDNA libraries derived from 28 kinds of tissues and cells, including seven tissues (brain, cerebellum, colon, hypothalamus, inguinal lymph node, ovary, and spleen) derived from pigs that were cloned from a sow subjected to genome sequencing. We obtained more than 330,000 EST reads from the 5'-ends of the cDNA clones. Comparison with human and bovine gene catalogs revealed that the ESTs corresponded to at least 15,000 genes. cDNA clones representing contigs and singlets generated by assembly of the EST reads were subjected to full-length determination of inserts. We have finished sequencing 31,079 cDNA clones corresponding to more than 12,000 genes. Mapping of the sequences of these cDNA clones on the draft sequence of the pig genome has indicated that the clones are derived from about 15,000 independent loci on the pig genome., Conclusions: ESTs and cDNA sequences derived from full-length-enriched libraries are valuable for annotation of the draft sequence of the pig genome. This information will also contribute to the exploration of promoter sequences on the genome and to molecular biology-based analyses in pigs.

The number of immunoglobulin heavy chain (IGH) constant genes (IGHC) varies among mammals. To annotate the porcine IGHC genes, we sequenced the entire IGHC-containing genomic region from a single porcine haplotype. The resulting contiguous sequence included in 5' the IGH diversity (D) gene cluster and in 3' TMEM121, which flank the IGHC cluster in the human genome, suggesting that we had obtained the entire genomic region containing porcine IGHC. This region was about 190-kb long, in good agreement with those of other mammals. The porcine IGHC cluster contained 10 genes, IGHM, IGHD, six IGHG genes, IGHE and IGHA. The porcine IGHG genes formed a cluster between IGHD and IGHE, with IGHG3 considered as the most ancient IGHG gene, located at the beginning of the IGHG cluster. Furthermore, the porcine sequence contained two IGHG5 and two IGHG6 genes, but no IGHG genes for IgG2 and IgG4, suggesting flexibility within the IGHG cluster. We also recorded structural differences in the switch regions of the IGHC genes that may be important in their transcription. This haplotype can serve as a reference for future studies on other haplotypes and for functional analysis of porcine immunoglobulin (IG) isotypes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Nucleotide oligomerization domain 2 (NOD2) is a cytosolic pattern recognition receptor (PRR) that responds to muramyldipeptide (MDP), a component of peptidoglycans of gram positive and negative bacteria. NOD2 is involved in the modulation of signaling pathways for other PRRs, such as Toll-like receptors. Polymorphisms in NOD2 may evoke bowel disorders, and human Crohn's disease is significantly correlated with mis-sense insertion of the NOD2 gene. Such polymorphisms affecting ligand recognition in the NOD2 gene may also influence bowel flora in livestock, which is compromised by bowel diseases such as diarrhea. We investigated the functional variance of mis-sense polymorphisms in ligand recognition by porcine NOD2. The 1949T>C polymorphism, located in the region encoding the hinge domain of the molecule, notably diminished the functional response of porcine NOD2 to MDP. By comparison, the 2197A>C polymorphism, localized in the region corresponding to leucine-rich repeats, significantly augmented the response of porcine NOD2 to the ligand. The 1949C allele was rare among pig breeds, suggesting that this mutation is a disadvantage to pigs in their immune response to microbes. The 2197C allele, in contrast, was widely distributed among Western breeds and is most likely to be derived from wild boars in Asia. This is the first report of a causal relationship between molecular function and polymorphisms in PRRs in non-primate, non-rodent mammals. These findings suggest that the 2197C allele might confer an immune response advantage in modern pig breeds and may be a useful marker for breeding aimed at disease resistance in pigs.

Pathogens localized extracellularly or incorporated into endosomes are recognized mainly by Toll-like receptors, whereas pathogens and pathogen-derived molecules that invade into the cytoplasm of host cells typically are recognized by intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene (RIG)-like helicases (RLHs) and nucleotide-binding oligmerization domain (NOD)-like receptors (NLRs). RIG-I and melanoma differentiation-associated gene 5 (MDA5), which belong to the RLH family, recognize viral genomic RNA, whereas NOD2, a member of the NLR family, responds to microbial peptidoglycans. These receptors may play an important role in pig opportunistic infectious diseases, such as pneumonia and diarrhea, which markedly impair livestock productivity, such that polymorphisms of these receptor genes are potential targets of pig breeding to increase disease resistance. Here, we report single nucleotide polymorphisms (SNPs) in porcine DDX58, IFIH1, and NOD2, which encode RIG-I, MDA5, and NOD2, respectively. Interestingly, compared with DDX58 and IFIH1, NOD2 abounded in nonsynonymous SNPs both throughout the coding sequence and in sequences encoding domains important for ligand recognition, such as helicase domains for RIG-I and MDA5 and leucine-rich repeats in NOD2. These differences in the distribution of SNPs in intracellular PRRs may parallel the diversity of their ligands, which include nucleic acids and peptidoglycans.

Comparative mapping studies facilitate the identification of genes located in quantitative trait locus (QTL) regions in domestic animals by utilizing information from the human genome. Radiation hybrid (RH) mapping is effective for this purpose because of its high resolution in ordered gene mapping on chromosomes. We constructed an RH map of pig chromosome 7, by adding 23 markers associated with genes. This RH map clearly demonstrated the mosaic of homology between pig chromosome 7 (SSC7) and human chromosomes 6, 14 and 15 at a 'gene' level, and was confirmed by linkage analysis. Clarification of the homology of SSC7 to human chromosomes will contribute to the elucidation of the gene(s) responsible for QTL detected on this chromosome.