H. Hiraiwa

Cloning and Characterization of the Gene for a New Epithelial β-Defensin GENOMIC STRUCTURE, CHROMOSOMAL LOCALIZATION, AND EVIDENCE FOR ITS CONSTITUTIVE EXPRESSION

Mammalian β-defensins are endogenous cysteine-rich peptide antibiotics that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine β-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans ∼1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15.1 within a region of conserved synteny to the chromosomal locations of human and mouse α- and β-defensins. We also provide several independent lines of evidence showing that thepBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial β-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-α, and interleukin (IL)-1β failed to up-regulate the expression ofpBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium orActinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1β. The constitutive expression ofpBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-κB or NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.

Genomic structure around joining segments and constant regions of swine T‐cell receptor α/δ (TRA/TRD) locus

A complete genomic region of 131·2 kb including the swine T‐cell receptor α/δ constant region (TRAC/TRDC) and joining segments (TRAJ/TRDJ) was sequenced. The structure of this region was strikingly conserved in comparison to that of human or mouse. All of the 61 TRAJ segments detected in the human genomic sequence were detected in the swine sequence and the sequence of the protein binding site of T early alpha, the sequence of the α enhancer element and the conserved sequence block between TRAJ3 and TRAJ4 are highly conserved. Insertion of the repetitive sequences that interspersed after the differentiation of the species in mammals such as short interspersed nucleotide elements is markedly suppressed in comparison to other genomic regions, while the composition of the mammalian‐wide interspersed sequences is relatively conserved in human and swine. This observation indicates the existence of a highly selective pressure to conserve this genomic region around TRAJ throughout the evolution of mammals.

Assignment of 280 swine genomic inserts including 31 microsatellites from BAC clones to the swine RH map (IMpRH map)

Abstract. A precise genetic map containing anonymous markers and genes is indispensable for the efficient selection of candidate gene(s) responsible for quantitative trait loci (QTL) traits. For this purpose, a first version of a radiation hybrid cell (RH) map has been constructed by using the INRA-University of Minnesota RH panel for 757 markers (IMpRH) (Hawken et al. 1999, Mamm. Genome 10: 824–830). In this study, 280 swine genomic fragments in BAC clones were assigned to the IMpRH map; 255 BAC clones were successfully linked to first-generation linkage groups (LOD > 4.8). The remaining 25 clones could not be mapped, because their lod-scores to the closest markers in the first generation map were less than 4.8. In addition, 16 BAC clones, mapped to swine Chromosome (Chr) 1 by IMpRH mapping, were subjected to isolation of microsatellites (MSs). Thirty-one MSs were isolated from 15 BAC clones, and 24 of 31 (77%) MSs derived from 14 clones were found to be polymorphic. We also mapped both termini of 12 BAC clones to the IMpRH map, in order to measure resolution of the IMpRH map; the resolution was found to range from 8 kb/centiRay to more than 126 kb/centiRay depending on the region.

Genomic organization and assignment of the interleukin 7 gene (IL7) to porcine chromosome 4q11→q13 by FISH and by analysis of radiation hybrid panels

Interleukin 7 (IL7) is a cytokine that has many immunological functions, including regulation of hematopoiesis and peripheral lymphocytes. cDNA and a genomic DNA segment containing the porcine IL7 gene were isolated and sequenced, showing that porcine IL7 consists of 176 amino acids and that its gene spans over about 13 kb of genomic DNA. Porcine IL7 has 85% and 73% homology with human IL7 in terms of the nucleotide and amino acid sequences, respectively. Whereas the murine IL7 gene does not have an exon corresponding to human exon 5 (Lupton et al., 1990), the porcine IL7 gene was found to contain the same exon-intron structure as the human gene. These findings, together with the upstream structure of the cDNA elucidated in the present study, indicate that the relationship between swine and human IL7 is closer than that between mouse and human IL7. The IL7 gene was mapped to swine chromosome 4q11→q13 by fluorescence in situ hybridization and, using a radiation hybrid panel, was localized between microsatellite markers Sw1336 and Sw1073 on the same chromosome.

Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse.

Bi- and uni-directional chromosome painting (ZOO-FISH) and gene mapping have revealed correspondences between human chromosome (HSA) 17 and porcine chromosome (SSC) 12 harboring economically important quantitative trait loci. In the present study, we have assigned 204 genes localized on HSA17 to SSC12 to generate a comprehensive comparative map between HSA17 and SSC12. Two hundred fifty-five primer pairs were designed using porcine sequences orthologous with human genes. Of the 255 primer pairs, 208 (81.6%) were used to assign the corresponding genes to porcine chromosomes using the INRA-Minnesota 7000-rad porcine × Chinese hamster whole genome radiation hybrid (IMpRH) panel. Two hundred three genes were integrated into the SSC12 IMpRH linkage maps; and one gene, PPARBP, was found to link to THRA1 located in SSC12 but not incorporated into the linkage maps. Three genes (GIT1, SLC25A11, and HT008) were suggested to link to SSC12 markers, and the remaining gene (RPL26) did not link to any genes/expressed sequence tags/markers registered, including those in the present study. A comparison of the gene orders among SSC12, HSA17, and mouse chromosome 11 indicates that intra-chromosomal rearrangements occurred frequently in this ancestral mammalian chromosome during speciation.

A dense comparative gene map between human chromosome 19q13.3→q13.4 and a homologous segment of swine chromosome 6

The human chromosome (HSA)19q region has been shown to correspond to swine chromosome (SSC) 6q11→q21 by bi-directional chromosomal painting and gene mapping. However, since the precise correspondence has not been determined, 26 genes localized in HSA19q13.3→q13.4 were assigned to the SSC6 region mainly by radiation hybrid (RH) mapping, and additionally, by somatic cell hybrid panel (SCHP) mapping, and fluorescent in situ hybridization (FISH). Out of the 26 genes, 24 were assigned to a swine RH map with LOD scores greater than 6 (threshold of significance). The most likely order of the 24 genes along SSC6 was calculated by CarthaGene, revealing that the order is essentially the same as that in HSA19q13.3→q13.4. For AURKC and RPS5 giving LOD scores not greater than 6, SCHP mapping and FISH were additionally performed; SCHP mapping assigned AURKC and RPS5 to SSC6q22→q23 and SSC6q21, respectively, which is consistent with the observation of FISH. Consequently, all the genes (26 genes) examined in the present study were shown to localize in SSC6q12→q23, and the order of the genes along the chromosomes was shown to be essentially the same in swine and human, though several intrachromosomal rearrangements were observed between the species.

Cloning and characterization of porcine common γ chain gene

The common γ chain, which was originally identified as a component of interleukin-2 receptors (IL-2R), plays a key role in differentiation of T lymphocytes and natural killer (NK) cells. In the present study, cDNA of the porcine common γ chain gene and its genomic DNA were molecularly cloned and characterized. The porcine common γ chain gene was found to consist of 8 exons, spanning approximately 3.7 kb, and to encode a 368-amino acid polypeptide. The amino acid sequence showed 82.4%, 71.1%, 86.1%, and 84.8% similarities with that of human, murine, bovine, and canine chains, respectively. The common γ chain gene was assigned to swine chromosome Xq13 by FISH analysis and was consistent with the result of radiation hybrid (RH) mapping. When various porcine tissues were examined for the expression of this gene, the expression was observed in lymphocytes and lymphocyte-related tissues. Since GATA, T cell factor-1 (TCF-1), Ets-1, activated protein2 (AP-2), and Ikaros2 binding motifs were demonstrated in the 5′...

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11

We are constructing high-resolution, chromosomal ‘test’ maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH212,000-rad)to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH212,000-rad panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH7,000-rad panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH12,000-rad FW maps, respectively, with an accumulated map distance of 4046.5 cR12,000 and 1355.2 cR7,000 for SSC2p, and 4244.1 cR12,000 and 1802.5 cR7,000 for SSC9p. The kb/cR ratio in the IMNpRH212,000-rad FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH7,000-rad FW maps was 47.1 and 36.3, respectively, or an ∼3.0-fold increase in map resolution in the IMNpRH12,000-rad panel over the IMpRH7,000-rad panel. The integrated IMNpRH12,000-rad andIMpRH7,000-rad maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.

Assignment of 101 genes localized in HSA10 to a swine RH (IMpRH) map to generate a dense human-swine comparative map

Economically important traits such as growth and backfat in pigs have been shown to be influenced by genes in swine chromosome (SSC) 10q12→qter corresponding to human chromosome (HSA) 10p. However, since gene information in the swine chromosomal region was limited, we attempted to generate a dense comparative map between SSC10 and HSA10 by mapping the 115 genes of HSA10 to a swine RH map (IMpRH map). In the mapping ten genes were assigned to SSC10, 88 to SSC14, and one to SSC3. One gene was suggested to link to SSC3, and another to SSC9. The correspondences between HSA10 and SSC10 and between HSA10 and SSC14 were essentially consistent with the observations obtained from bi/uni-directional chromosome painting or other results. This study further indicated that a large number of intrachromosomal rearrangements occurred in the synteny-conserved regions following species separation.

A high-resolution comparative map of porcine chromosome 4 (SSC4)

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.