Sequence of the pig major histocompatibility region containing the classical class I genes

A segment comprising 307,078 nucleotides of the pig major histocompatibility complex (SLA) was completely sequenced. The segment corresponded to the entire SLA classical class I-containing region of the serologically defined SLA H01 haplotype. In all, 11 genes were characterized, comprising 7 class I genes located on the centromeric part of the sequence (SLA-1, 2, 3, 4, 5, 9, and 11) and 4 ring finger-related family genes located on its telomeric part. No member of one family was intermingled with a member of the other or with any third-party gene. All class I genes except SLA-11 were similarly orientated. The SLA-1, 2, and 3 genes displayed both promoter and overall coding regions compatible with normal functions. The SLA-4, 11, and 9 genes were considered pseudogenes because they exhibited marked anomalies. Although the SLA-5 gene had a complete coding region, it displayed mutations in promoter elements which could modify its expression. The great molecular similarity observed among the class I genes extended far outside them, and resulted from segmental duplications. The ring finger genes exhibited great homology with their human counterparts. In pig, one of these genes appeared to correspond to a complete gene which in humans is probably a pseudogene. In all, the 11 genes characterized span about 20% of the total sequence. The remaining 80% consists of interspersed repeat elements. The present results, together with the sequence previously reported involving the SLA class I-related genes, open the way for a better understanding of pig MHC organization.     

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     

Spatial arrangement of pig MHC class I sequences

Bacterial artificial chromosome (BAC) clones were assigned within the pig major histocompatibility complex (Mhc) by polymerase chain reaction-screening and Southern blot hybridization using sequence-tagged site (STS) markers and BAC end-rescued sequences. In all, 35 BAC clones were discovered containing 12 anchor genes of the SLA class I region and two genes of the SLA class III region. Twenty of these 35 clones comprised two distinct class I gene clusters, each spanning about 100 kilobases. One cluster enclosed three class I related genes (SLA-6 to -8) and two genes (MIC-1 and MIC-2) more distantly related to class I. The other cluster enclosed typical class I genes, of which three (SLA-1, -2, and -3) were transcribed by fibroblasts homozygous for the H01 haplotype which we used to construct a pig BAC library. Ordered clones are certainly helpful in isolating agronomically, biologically, and medically important genes. They would also be useful for inducing genetic modifications in pig cell lines.     

Uterine MHC class I molecules and beta 2-microglobulin are regulated by progesterone and conceptus interferons during pig pregnancy

MHC class I molecules and beta(2)-microglobulin (beta(2)m) are membrane glycoproteins that present peptide Ags to TCRs, and bind to inhibitory and activating receptors on NK cells and other leukocytes. They are involved in the discrimination of self from non-self. Modification of these molecules in the placenta benefits pregnancy, but little is known about their genes in the uterus. We examined the classical class I swine leukocyte Ags (SLA) genes SLA-1, SLA-2, and SLA-3, the nonclassical SLA-6, SLA-7, and SLA-8 genes, and the beta(2)m gene in pig uterus during pregnancy. Uterine SLA and beta(2)m increased in luminal epithelium between days 5 and 9, then decreased between days 15 and 20. By day 15 of pregnancy, SLA and beta(2)m increased in stroma and remained detectable through day 40. To determine effects of estrogens, which are secreted by conceptuses to prevent corpus luteum regression, nonpregnant pigs were treated with estradiol benzoate, which did not affect the SLA or beta(2)m genes. In contrast, progesterone, which is secreted by corpora lutea, increased SLA and beta(2)m in luminal epithelium, whereas a progesterone receptor antagonist (ZK137,316) ablated this up-regulation. To determine effects of conceptus secretory proteins (CSP) containing IFN-delta and IFN-gamma, nonpregnant pigs were implanted with mini-osmotic pumps that delivered CSP to uterine horns. CSP increased SLA and beta(2)m in stroma. Cell-type specific regulation of SLA and beta(2)m genes by progesterone and IFNs suggests that placental secretions control expression of immune regulatory molecules on uterine cells to provide an immunologically favorable environment for survival of the fetal-placental semiallograft.     

Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations

The highly polymorphic swine leucocyte antigen ( SLA ) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation success. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to type alleles at the three classical SLA class I loci ( SLA-1 , SLA-3 and SLA-2 ) using the PCR-sequence-specific primer (PCR-SSP) strategy. This typing system relies on 47 discriminatory PCR primer pairs designed to amplify the SLA class I alleles by groups that have similar sequence motifs. We applied this low-resolution group-specific typing method to characterize the SLA class I alleles present in three outbred pig populations ( n =  202). Alleles from 24 class I allele groups corresponding to 56 class I genotypes were detected. We also identified 23 low-resolution SLA class I haplotypes in these pigs and found haplotypes Lr-1.0 ( SLA-1 *01XX- SLA-3 *01XX- SLA-2 *01XX) and Lr-4.0 ( SLA-1 *04XX- SLA-3 *04XX- SLA-2 *04XX) in all three pig populations with a high prevalence. Over 80% of the pigs examined ( n  =   162) were found to bear at least one of these haplotypes, resulting in a combined haplotype frequency of nearly 50%. This PCR-SSP-based typing system demonstrates a reliable and unambiguous detection of SLA class I alleles, and can be used to effectively investigate the SLA diversity in outbred pig populations. It will help to identify the role of SLA antigens in disease-resistant pigs and may facilitate the development of effective vaccines.     

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.     

The porcine Major Histocompatibility Complex and related paralogous regions: a review

The physical alignment of the entire region of the pig major histocompatibility complex (MHC) has been almost completed. In swine, the MHC is called the SLA (swine leukocyte antigen) and most of its class I region has been sequenced. Over one hundred genes have been characterised, including the classical class I and class I-related genes, as well as the class II gene families. These results in swine provide new evidence for the striking conservation during the evolution of a general MHC framework, and are consistent with the location of the class I genes on segments referred to as permissive places within the MHC class I region. Recent results confirm the involvement of the SLA region in numerous quantitative traits.     

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.     

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.     

Isolation of four HSP70 genes in the pig and localization on Chromosomes 7 and 14

For insight into the general organization of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), four sequences related to the heat-shock proteins HSP70 were characterized by screening of a pig genomic cosmid library with a swine cDNA HSP70 2.6-kb probe. This yielded three positive clones: HC2.2, HC3.2, and HC4.2. Restriction site maps revealed a large overlap of HC2.2 with HC3.2, whereas HC4.2 was independent. Southern blot hybridization with the 5 section, the central section, and the 3 section of the 2.6-kb probe and also with a swine 4.5-kb HSP70 genomic probe suggested the existence, within the overlapping clones, of three distinct HSP70 sequences encompassing a segment no longer than 22 kb. The HC4.2 clone, which hybridized with the same probes, displayed a single band of 7.3 kb, probably corresponding to one gene only. Fluorescent in situ hybridization on swine chromosome metaphases with the whole HC2.2 or HC4.2 cosmids allowed the assignment of HC2.2 to MHC region on Chromosome (Chr) 7 (Cen-p1.1), and of HC4.2 to Chr 14 (q2.4–2.5). Thus, as in humans, the swine MHC comprises three closely linked HSP70 loci. The presence of additional genes belonging to the same inducible HSP70 gene family can be expected from what is known in humans. The HSP70 gene found here on the pig Chr 14 may be one of these putative unidentified genes.     

Mapping of the SLA complex class III region by pulsed field gel electrophoresis

The fine order of genes in the class III region of the swine major histocompatibility complex (MHC), the SLA complex, was examined by pulsed field gel electrophoresis (PFGE) and Southern blot analysis. Four genes, C2, HSP70, TNF, and CYP21, were analyzed. The CYP21, C2, and HSP70 genes were all located within a 200-kb NotI fragment. The C2, HSP70, and TNF genes cohybridized to a 420-kb SalI fragment. The TNF gene is linked to the class I region by a 390-kb NotI fragment. Combined with a previous study from our lab, the order of genes in the SLA complex is class II-class III [(CYP21/C4)-(Bf/C2/HSP70)-TNF]-class I. The size of the class III region from CYP21 to TNF is estimated to be 500 kb. This size and the order of the genes in the swine class III region are similar to those of human, mouse, goat, and rabbit, which confirms the high conservation of class III gene organization across species.     

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.     

Molecular characteristics of the SLA-2 gene from Chinese Hebao pigs

To study the molecular characteristics of swine leukocyte antigen (SLA) class I from the Hebao pig, a rare inbreed in China, a pair of primers was designed to amplify the SLA-2 gene (SLA-2-HB) and then the molecular characteristics of the gene were analyzed by computer. After cloning, sequencing and computer analysis, four SLA-2-HB alleles were found, all of 1119 bp. Sites 3-1097 were an open reading frame encoding 364 amino acids with two sets of intra-chain disulfide bonds comprising four cysteines situated in sites 125, 188, 227 and 283. By alignment of SLA-2-HB sequences with other SLA-2 alleles in the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank database, nine key variable amino acid sites were found in the extracellular domain of the SLA-2-HB alleles at sites 23(F), 24(I), 43(A), 44(K), 50(Q), 73(N), 95(I), 114(R) and 216(S), which could be used to differentiate other SLA-2 alleles. The amino acid identities between SLA-2-HB and other SLA-2, SLA-3 and SLA-1 alleles were 87.1-97.0%, 85.0-93.9% and 83.3- 88.6%, respectively. The phylogenetic tree of SLA-2-HB showed that it was relatively independent of the other SLA-2 genes. Furthermore, the SLA-2-HB alleles were similar to HLA-B15 and HLA-A2 functional do- mains and preserved some functional sites of HLA-A2. It was concluded that SLA-2-HB is an allele of SLA-2 and that the Hebao pig might have evolved independently in China.     

Analyzing the genetic characteristics and function of the swine leukocyte antigen 2 gene in a Chinese inbreed of pigs

To study the genetic characteristics and function of swine leukocyte antigen (SLA) class I from the Hebao pig, a rare inbreed in China, a pair of primers was designed to amplify the SLA-2 gene (SLA-2-HB) and then the genetic characteristics of the gene were analyzed. The 3D homology modeling was used to analyze the structure and function of SLA-2-HB proteins. After cloning, sequencing and computer analysis, four SLA-2-HB alleles were found, all of 1119 bp. Sites 3-1097 were an open reading frame encoding 364 amino acids with two sets of intra-chain disulfide bonds comprising four cysteines situated in sites 125, 188, 227 and 283. By alignment of SLA-2-HB sequences with other SLA-2 alleles in the IPD database, 11 key variable amino acid sites were found in the extracellular domain of the SLA-2-HB alleles at sites 23(F), 24(I), 43(A), 44(K), 50(Q), 73(N), 95(I), 114(R), 155(G), 156(E) and 216(S), which could be used to differentiate other SLA-2 alleles. The 3D homology modeling demonstrated that the eight of 11 key variable amino acid sites were all in antigenic binding groove of SLA-2-HB proteins. The amino acid identities between SLA-2-HB and other SLA-2, SLA-1 and SLA-3 alleles were 86.2-97.0%, 85.0-93.9% and 83.3-88.6%, respectively. The phylogenetic tree of SLA-2-HB showed that it was relatively independent of the other SLA-2 genes. Furthermore, the SLA-2-HB alleles were similar to HLA-B15 and HLA-A2 functional domains and preserved some functional sites of HLA-A2. It was concluded that SLA-2-HB are novel alleles of SLA-2 and that the Hebao pig might have evolved independently in China.     

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

Background

Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.

Results

The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.

Conclusion

The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.