Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL, and GenBank databases under accession numbers AB158486 and AB158487     

Structure and expression of two porcine genomic clones encoding class I MHC antigens

Two nonallelic porcine class I MHC (SLA) genes have been isolated and characterized. Both genes are expressed in mouse L cells, directing the synthesis of class I SLA molecules that carry common monomorphic determinants but are serologically distinct. The corresponding DNA sequences have been determined. The organization of both of these genes is similar to that of other class I genes: a leader exon, three exons encoding extracellular domains, a transmembrane exon, and three intracytoplasmic exons. The two genes are highly homologous in both exon and intron segments, with average homologies of 88% and 80%, respectively. Nucleotide changes in exon 2 are clustered, whereas those in the other exons are dispersed throughout. Comparison of the swine DNA sequences with class I genes from other species reveals a generally high conservation of exons 2, 3, 4, and 6 with lower homology in the remaining protein-encoding domains. Introns are markedly less well conserved, although moderate homology is found between swine and human class I MHC genes in both introns and 3' flanking regions. Taken together with comparisons of the deduced protein sequences, these data indicate an order of swine greater than human greater than rabbit greater than mouse in the relationship of class I genes.     

Difference in number of loci of swine leukocyte antigen classical class I genes among haplotypes

The structure of the entire genomic region of swine leukocyte antigen (SLA)-the porcine major histocompatibility complex--was recently elucidated in a particular haplotype named Hp-1.0 (H01). However, it has been suggested that there are differences in the number of loci of SLA genes, particularly classical class I genes, among haplotypes. To clarify the between-haplotype copy number variance in genes of the SLA region, we sequenced the genomic region carrying SLA classical class I genes on two different haplotypes, revealing increments of up to six in the number of classical class I genes in a single haplotype. All of the SLA-1(-like) (SLA-1 and newly designated SLA-12) and SLA-3 genes detected in the haplotypes thus analyzed were transcribed in the individual. The process by which duplication of SLA classical class I genes was likely to have occurred was interpreted from an analysis of repetitive sequences adjacent to the duplicated class I genes.     

Mapping of the SLA complex of miniature swine: mapping of the SLA gene complex by pulsed field gel electrophoresis.

The overall order of the regions of the swine major histocompatibility complex (MHC), the SLA complex, was determined by pulsed field gel electrophoresis (PFGE). It was found that the order of the regions is class II-class III-class I. A class I probe hybridized to a 420 kb Mlu I and a 420 kb Not I fragment as did a class III probe for C2. None of the class II probes hybridized to these fragments. Thus, linkage of class I to class III was shown. The class III C2, Bf, and C4 genes were found to residue in a 190 kb Not I fragment. Linkage of class III and class II genes was shown when both the class III C4 and the class II DR probes hybridized to the same 195 kb Sac II and 340 kb Not I fragments. The class I probe did not hybridize to these fragments. The order of the regions, class II-class III-class I, is similar to that of human MHC genes and may have been conserved in evolution so that coordinated expression of MHC genes could be achieved.     

Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation

To lay background for studying rejection mechanisms in xenotransplantation and developing the strate-gies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced. The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors. All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their ac-cession numbers AY102467- AY102472 from GenBank. This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A^*0201) are better than those between pigs and mice (H-2D^b/H-2K^b). The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules. The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted. A functional test indicated that the human T cells specific for the prokaryotically expressed SLA Plprotein could respond quite well in vitro to the class I-positive swine chon-drocytes and PBMCs in presence of human APCs. This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens.     

Establishment of a resource population of SLA haplotype-defined Korean native pigs

The highly polymorphic porcine major histocompatibility complex (MHC), or the swine leukocyte antigens (SLA), has been repeatedly associated with variations in swine immune response to pathogens and vaccines as well as with production traits. The SLA antigens are also important targets for immunological recognition of foreign tissue grafts. We recently established a resource population of Korean native pigs as models for human transplantation and xenotransplantation research. In this study, 115 animals derived from three generations of the Korean native pigs were genotyped for three SLA class I (SLA-2, SLA-3 and SLA-1) and three SLA class II loci (DRB1, DQB1, DQA) using PCR with sequence-specific primers (PCR-SSP) at the allele group resolution. A total of seven SLA haplotypes (Lr-5.34, Lr-7.23, Lr-31.13, Lr-56.23, Lr-56.30, Lr-59.1, Lr-65.34), comprising six unique class I and five unique class II haplotypes, were characterized in the founding animals. Class I haplotype Lr-65.0 and class II haplotype Lr-0.34 were novel; and together with Lr-56.0 these haplotypes appeared to be breed-specific. In the progeny population, Lr-7.23 and Lr-56.30 appeared to be the most prevalent haplotypes with frequencies of 34.7% and 31.6%, respectively; the overall homozygosity was 27.4%. This resource population of SLA-defined Korean native pigs will be useful as large animal models for various transplantation and xenotransplantation experiments, as well as for dissecting the roles of SLA proteins in swine disease resistance and production traits.     

Marked genetic polymorphism of the swine steroid 21-hydroxylase gene, and its location between the SLA class I and class II regions

Restriction fragment length polymorphism (RFLP) analysis of the swine 21-hydroxylase (CYP21) region was conducted on 31 unrelated SLA class I typed pigs, mainly Large Whites, including 15 haplotypes. Ten haplotypes were from SLA genotypic homozygotes and five were from SLA class I phenotypic homozygotes. DNA digestion with Hin dIII, TaqI and PstI, and hybridization to a 4.5-kb swine CYP21 genomic probe yielded respectively two, four and three RFLP patterns. Six patterns were identified with combined RFLP. In addition, analysis of the CYP21 region in families comprising several SLA recombinants demonstrated that the CYP21 gene lies in the DNA segment between the SLA class I and class II regions. These overall results reinforce our previous conclusion about the existence in the pig of a single 21-hydroxylase gene. The characterization of at least six CYP21 allelic patterns provides a new tool for studying the associations between the SLA region and zootechnical traits.     

RFLP analysis of SLA haplotypes in Swiss large white and American Hampshire pigs using SLA class I and class II probes

Summary

Restriction fragment length polymorphism (RFLP) analyses of swine leukocyte antigen (SLA) class I and class II genes from Swiss Large White and American Hampshire families were performed using porcine DNA probes. Class I and class II RFLPs associated with the serologically‐defined haplotypes SLA H1, H8, H16 and H24 and with serotypes SLA 15, 16; SLA 14; and SLA 6, SB 19, were identified. Seven allelic class I RFLP patterns were observed. For genes in the SLA class II region, six allelic RFLP patterns of DQA and DQB; five allelic RFLP patterns of DRA; and seven allelic RFLP patterns of DRB were observed. The serologically‐defined H8 haplotype was subtyped based on differences in class II RFLPs.     

The Phylogenetic History of the MHC Class I Gene Families in Pig,Including a Fossil Gene Predating Mammalian Radiation

More than 990 kb of the 1200 kb in the SLA class I region of the pig major histocompatibility complex (MHC) have been sequenced. The present study was designed to establish the evolution of this region which was best understood by distinguishing three periods. The most recent period, which extended from 40 to 15 mya, probably corresponded to five rounds of duplication of a basic unit. This unit consisted of a single class I gene linked to widely dispersed repeats, and one SLA-specific repeat motif. The duplications gave rise to six SLA classical class I genes. The second evolutionary period corresponded to the emergence of the SLA nonclassical class I genes, i.e. after the suidae separated from the other artiodactyl species about 65 mya. The third period appeared to correspond to a much more remote age when the ancestor of the gene SLA-11 existed. Comparative studies of the human and pig sequences of the class I-containing segments indeed revealed the presence within the human HSR1-ZNF segment of relics of a human class I fossil gene which appeared to be orthologous to the 5 moiety of the SLA-11 pseudogene. This was the first evidence that a class I gene existed in this location at least 110-120 mya in the MHC class I region of the precursor of the mammalian species. Human/pig sequence comparison also revealed that the presumably functional pig MIC2 gene was probably orthologous to the human functional MICA or MICB genes.     

Mapping of the SLA complex of miniature swine: Mapping of the SLA gene complex by pulsed field gel electrophoresis

The overall order of the regions of the swine major histocompatibility complex (MHC), the SLA complex, was determined by pulsed field gel electrophoresis (PFGE). It was found that the order of the regions is class II-class III-class I. A class I probe hybridized to a 420 kbMlu I and a 420 kbNot I fragment as did a class III probe forC2. None of the class II probes hybridized to these fragments. Thus, linkage of class I to class III was shown. The class IIiC2, Bf, andC4 genes were found to reside in a 190 kbNot I fragment. Linkage of class III and class II genes was shown when both the class IIiC4 and the class IiDR probes hybridized to the same 195 kbSac II and 340 kbNot I fragments. The class I probe did not hybridize to these fragments. The order of the regions, class II-class III-class I, is similar to that of human MHC genes and may have been conserved in evolution so that coordinated expression of MHC genes could be achieved.     

Rapid assignment of swine leukocyte antigen haplotypes in pedigreed herds using a polymerase chain reaction-based assay

We present a simple assay to determine the swine leukocyte antigen (SLA) haplotypes of animals within two experimental populations of MHC defined miniature pigs. The Yucatan miniature pigs have four founder haplotypes ( w, x, y, z) and one recombinant haplotype ( q). The NIH miniature pigs have three founder haplotypes ( a, c, d) and two recombinant haplotypes ( f, g). Because most crossovers occur between the class I and class II regions, haplotypes can be assigned by typing one class I locus and one class II locus for practical purposes. We have previously characterized these seven founder haplotypes by sequencing the cDNA of three SLA class I loci, designated as SLA-1, SLA-3 and SLA-2 and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1 and SLA-DRB1. These sequences were used to design allele-specific primers to amplify one MHC class I and one MHC class II gene for each haplotype. Primers were tested for specificity in homozygous and heterozygous animals. Positive control primers were also designed to amplify a portion of the E-selectin or alpha-actin gene and multiplexed with the allele-specific primers to check for false negatives. This combination of allele-specific and positive control primers produced specific and robust PCR-site-specific primer assays for assigning SLA haplotypes in the two populations.     

Association of restriction fragment length polymorphisms of swine leucocyte antigen class I genes with production traits of Duroc and Hampshire boars

Summary. Restriction fragment length polymorphism analyses of swine leucocyte antigen (SLA) class I genes were performed on 70 Duroc and 38 Hampshire boars from the 1986-87 national performance tests of each breed in the USA. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA, isolated from white blood cells, using Pvu II endonuclease and a swine major histocompatibility complex (MHC) class I probe. Durocs had an average of 11 restriction fragments, with the most common being in 63% of the boars and the least common appearing in only one boar. Hampshire boars had an average of 12 restriction fragments, with the most common appearing in 73% of the boars and the least common appearing in only one boar. Least squares procedures and stepwise regression methods were used to examine the association between DNA restriction fragments and the selection index (INDEX), average daily gain (ADG), average backfat thickness (BF), loin muscle area (LEA), and age at 104kg (DAY104). In the Duroc breed one DNA restriction fragment was associated with decreased INDEX ( P < 0.05) and decreased ADG ( P < 0.05) whereas two other fragments were associated with increased BF ( P < 0.05). In the Hampshire breed two restriction fragments were associated with an increase in INDEX ( P < 0.05). Cluster analyses were used to group pigs of each breed on the basis of similar RFLP patterns. One cluster group in the Duroc breed was associated with lower average INDEX values ( P < 0.05), greater average DAY104 ( P < 0.05), and a larger mean LEA ( P < 0.05). In the Hampshire breed one cluster group was associated with lower INDEX ( P < 0.05). These results suggest there may be an association between swine MHC class I genes and performance traits in swine. The use of SLA class I restriction fragments, as genetic markers, may have potential in the future for improving pig performance.     

Association of restriction fragment length polymorphisms of swine leucocyte antigen class I genes with production traits of Duroc and Hampshire boars

Restriction fragment length polymorphism analyses of swine leucocyte antigen (SLA) class I genes were performed on 70 Duroc and 38 Hampshire boars from the 1986-87 national performance tests of each breed in the USA. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA, isolated from white blood cells, using PvuII endonuclease and a swine major histocompatibility complex (MHC) class I probe. Durocs had an average of 11 restriction fragments, with the most common being in 63% of the boars and the least common appearing in only one boar. Hampshire boars had an average of 12 restriction fragments, with the most common appearing in 73% of the boars and the least common appearing in only one boar. Least squares procedures and stepwise regression methods were used to examine the association between DNA restriction fragments and the selection index (INDEX), average daily gain (ADG), average backfat thickness (BF), loin muscle area (LEA), and age at 104 kg (DAY104). In the Duroc breed one DNA restriction fragment was associated with decreased INDEX (P less than 0.05) and decreased ADG (P less than 0.05) whereas two other fragments were associated with increased BF (P less than 0.05). In the Hampshire breed two restriction fragments were associated with an increase in INDEX (P less than 0.05). Cluster analyses were used to group pigs of each breed on the basis of similar RFLP patterns. One cluster group in the Duroc breed was associated with lower average INDEX values (P less than 0.05), greater average DAY104 (P less than 0.05), and a larger mean LEA (P less than 0.05). In the Hampshire breed one cluster group was associated with lower INDEX (P less than 0.05). These results suggest there may be an association between swine MHC class I genes and performance traits in swine. The use of SLA class I restriction fragments, as genetic markers, may have potential in the future for improving pig performance.     

猪主要组织相容性复合体研究进展

主要组织相容性复合体（major histocompatibility complex,MHC）分子首先作为主要组织相容性抗原被发现,进而以此命名。随着其抗原结合分子及T细胞信使生物学功能本质的揭示,关于MHC结构与功能的研究就进入了一个崭新时代,并在这一领域中取得了许多可喜的成果。论述了近年来猪MHC（SLA）结构与功能方面的研究进展,包括SLA的分类及多态性、SLA分子结构特点、SLA分子功能及SLA分子相关领域的最新研究进展。     

Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs

The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F ₂ descendants of F ₁ Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA‐1 , SLA‐2 , SLA‐3 ) and class II ( SLA‐DRB1 , SLA‐DQB1 , SLA‐DQA ) genes of 27 purebred Pietrain pigs using a combination of the high‐resolution sequence‐based typing (SBT) method and a low‐resolution (Lr) PCR‐based method using allele‐group, sequence‐specific primers (PCR‐SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr‐43.0 ( SLA‐1 *11XX– SLA‐3 *04XX– SLA‐2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr‐0.14 [ SLA‐DRB1 *0901– SLA‐DQB1 *0801– SLA‐DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr‐Pie‐0.1 [ SLA‐DRB1 *01XX/be01/ha04– SLA‐DQB1 *05XX– SLA ‐ DQA*blank] and Lr‐Pie‐0.2 [ SLA‐DRB1 *06XX– SLA‐DQB1 *03XX– SLA‐DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR‐SSP‐based assays represents a rapid and cost‐effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease‐resistant pigs in the context of SLA antigens to improve overall swine health.