Polymorphisms at position -308 in the promoter region of the TNF-alpha and in the first intron of the TNF-beta genes and spontaneous and lipopolysaccharide-induced TNF-alpha release in sarcoidosis.

TNF-alpha is a potent pro-inflammatory cytokine. Previous studies have proved that biallelic polymorphisms in the TNF-alpha (-308, TNFA) and TNF-beta genes (intron 1, TNFB) influence TNF-alpha production. In sarcoidosis, a chronic granulomatous disease, as a result of an unknown in vivo activation bronchoalveolar lavage (BAL) cells release high amounts of TNF-alpha, spontaneously and after in vitro stimulation. Thus, sarcoidosis could serve as a model to test the in vivo effect of TNF gene polymorphisms. We determined the TNFA and TNFB polymorphisms of 44 patients with sarcoidosis and found the following allele frequencies: 0.80, 0.20, 0.38 and 0.62 for TNFA1, TNFA2, TNFB1 and TNFB2, respectively. To examine the in vivo effect of the named polymorphisms on the TNF-alpha production, the spontaneous and LPS-induced TNF-alpha release of BAL cells and peripheral blood mononuclear cells were also determined in patients with sarcoidosis. Statistical analysis did not reveal any significant difference between sarcoidosis patients with different genotypes. The results show that TNFA and TNFB polymorphisms do not determine the level of TNF-alpha release of mononuclear cells activated during the course of sarcoid inflammation.     

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.     

Frequencies of the tumor necrosis factor gene polymorphisms in the Korean population

Tumor necrosis factor (TNF), a potent immuno-modulator and pro-inflammatory cytokine, has been implicated in many pathological processes. The TNFA and the TNFB genes, which encode TNFalpha and TNFbeta, respectively, are both located on the short arm of chromosome 6 between the class I and class II regions of the HLA complex. A striking feature of the entire HLA complex is a high degree of genetic variation. Two biallelic polymorphisms in the TNFA (- 308G/A) and TNFB (+ 252A/G) genes have been reported to be associated with TNF production and with susceptibility to inflammatory diseases. Population information on polymorphisms is essential for the study of genetic diseases. The aim of this study is to obtain accurate information about polymorphisms in the TNF genes in the Korean population. Allele frequencies of TNFA (- 308G/A) and TNFB (+ 252A/G) were measured in 581 unrelated Korean individuals by PCR-RFLP. Allele frequencies of each polymorphism were determined and compared with those previously reported in other populations. A significant difference was found for the allele frequencies of TNFA and TNFB gene in Koreans compared with Europeans. The - 308/A allele in the TNFA gene was very rare in Asians (0.008-0.096). The frequency of the - 308/A allele in Koreans was considerably lower than in Europeans (0.120-0.189). Contrary to lower frequency of the -308/A allele, that of + 252/G allele in the TNFB gene was higher than in Koreans (0.445) compared with Europeans (0.29-0.39). The polymorphisms and allele frequencies obtained in this study will be useful for genetic studies of common inflammatory diseases.     

Assignment of the porcine major histocompatibility complex to chromosome 7 by in situ hybridization

The major histocompatibility complex (SLA) of the domestic pig (Sus scrofa) was regionally mapped to 7p12----q12 by in situ hybridization with an SLA class I-specific recombinant DNA probe. This localization contradicts linkage data suggesting a possible assignment of the SLA locus to porcine chromosome 15.     

Assignment of the porcine aminopeptidase N (PEPN) gene to chromosome 7cen----q21.

The porcine aminopeptidase N (PEPN) gene was shown to be syntenic with the porcine major histocompatibility complex (SLA) using Southern blot analysis of a pig x mouse hybrid cell panel. Regional localization of the PEPN gene to 7cen----q21 was performed by in situ hybridization. The results suggest that synteny of the genes for mannose phosphate isomerase and PEPN is conserved in pig and man.     

猪白细胞Ⅱ类抗原基因多态性研究进展

本文对SLAⅡ类基因的染色体图谱定位、分子结构、基因分型及其多态性的研究进展作了介绍。重点介绍了SLAⅡ类基因的RFLP基因分型及其多态性研究。猪的主要组织相容性复合物Ⅱ类抗原基因座位存在不同程度的等位基因多态性。RFLP技术是一种简便而快捷的SLAⅡ类等位基因分型方法。SLAⅡ类基因的多态性和抗原肽结合位点密切相关,这些位点能结合处理过的抗原,然后递呈给T细胞。另外,在编码β1链的SLA-DRB1基因和SLA-DQB基因中有4个富含GC序列区。 Abstract:This article gave a detailed introduction about regional map,molecular structure,genotyping and polymorphism of SLA class Ⅱ genes.The pig major histocompatibility complex (MHC) class Ⅱ antigens have been known to exhibit a different degree of allelic polymorphism.The locus-specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA class Ⅱ from genomic DNA.SLA class Ⅱ polymorphism was related to the antigenic peptide binding sites.Detailed analysis of sequences showed that there were 4 GC-rich sequences in exon 2 of SLA-DQB and SLA-DRB1 genes.     

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     

A physical map including a new class I gene (cda12) of the human major histocompatibility complex (A2/B13 haplotype) derived from a monosomy 6 mutant cell line

To avoid interpretative problems due to restriction fragment length polymorphisms, the monosomy 6 mutant cell line BM19.7 was employed to establish a molecular map of the human major histocompatibility (HLA) complex in the A2,B13,Bw4,DRw6,DRw52,DQw1,DPw2 haplotype. Results were obtained mainly by field-inversion gel electrophoresis and Southern blotting techniques. The map extends to 4800 kb and includes the HLA complex with a length of 4200 kb. Five HTF islands could be positioned on the map. The class I region has a size of about 2000 kb and includes nonclassical HLA class I genes, some of which must be localized within 200 kb telomeric of HLA-A. A new class I gene, cda12, distinct from HLA-A, HLA-B, or HLA-C, has been localized within 50 kb from HLA-A. The class I region contains a gap of about 500 kb, just telomeric of HLA-C, in which further class I genes could not be detected. The class II region has a size of 1000 kb, which is separated from the class I region by about 1200 kb. The 5' end of the HLA-B gene is situated centromeric, giving an orientation opposite to that of the TNFA and TNFB loci. The estimated length of the HLA complex correlates well with its size determined cytogenetically using mutant cell lines with interstitial deletions.     

猪白细胞抗原复合体Ⅰ类和Ⅱ类研究进展

猪主要组织相容性复合体又称猪白细胞抗原复合体（SLA）,是猪基因组中基因密度最高的区域之一,也是多态性最高的区域。许多研究表明SLA在抗原提呈及免疫调节等方面起着重要的作用,其基因及基因组学研究在以猪为替代模型的人-猪器官移植、癌症、变态反应、猪的生产数量性状及对感染性疾病的应答、疫苗研制等方面都是热点。我们就目前Ⅰ类和Ⅱ类SLA分子功能基因多态性及与机体免疫水平、抗病育种和生产性状的相关性作一综述。     

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.     

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。     

Assignment of the major histocompatibility complex to the p1.4----q1.2 region of chromosome 7 in the pig (Sus scrofa domestica L.) by in situ hybridization

The pig major histocompatibility complex (SLA) was mapped by in situ hybridization. The probe used was a human cDNA containing most of the coding sequence for HLA-B7. The SLA complex was localized to the region p1.4----q1.2 on chromosome 7.     

Map arrangement of the SLA chromosomal region and the J and C blood group loci in the pig

Linkage studies of three-point crosses (triple backcross matings) showed that the linear sequence of three of the pig's immunogenetic traits — the SLA major histocompatibility complex and the J and C blood group loci — is SLA-J-C . Andresen & Baker (1964) and Rasmusen (1965) described close linkage between the J and C blood group loci and respectively found their recombination frequency to be 5.29 ± 1.1 % and 7.00 ± 3.4 %; by combining the data the exact frequency was determined at 5.75 ± 0.79 % (Muir & Rasmusen, 1974). Later, linkage of the SLA major histocompatibility complex with both J (Hruban et al., 1976) and C (Hruban et al., 1977) erythrocytic loci was found. The maximum tabular lod score values were found in the recombination fraction Θ= 0.10 in comparison of SLA and J and in the fraction Θ= 0.20 in comparison of SLA and C (Hruban et al., 1977).     

Cloning, transcription and chromosomal localization of the porcine whey acidic protein gene and its expression in HC11 cell line

The whey acidic protein (WAP) is the major whey protein of rodent, rabbit and camel. Recently, it was identified in the milk of swine (Simpson et al., 1998. J. Mol. Endocrinol. 20, 27-35). In this paper, the cloning of the pig WAP cDNA and of bacterial artificial chromosome (BAC) construct containing the entire porcine WAP gene is reported. The comparison of the coding sequence of the pig WAP gene to rodent or lagomorph WAP sequence already published demonstrated that only exon sequences are partially conserved. The porcine WAP gene was localized on the subtelomeric region of the chromosome 18. The estimation of the expression of the swine WAP gene in the mammary gland from lactating animals revealed a high level of expression. In order to compare the expression level of the porcine WAP gene from the large genomic fragment which contained 70 kb downstream and 50 kb upstream the pig WAP gene or the smaller one (1 kb downstream and 2.4 kb upstream), these two genomic fragments were transfected in HC11 cell line. The BAC construct was expressed 15 times higher than the plasmid when reported to the integrated copy number. This report suggests that the HC11 cell line is a useful tool to identify the regulatory sequences of milk protein genes.     

Cloning and sequencing of the porcine complement factor B

A genomic clone, SSBf1, containing the complement factor B (BF), a major histocompatibility class III antigen, has been isolated from a porcine genomic library. Partial sequencing and comparison with a human BF gene has identified seven exons coding for amino acids of Ba and Bb, the two subunits of BF. The protein sequence similarity with the human BF is on the average 87%. Southern blot analysis confirmed the existence of only one BF gene per haploid genome. Restriction fragment length polymorphism typing with Taq I showed that there are at least three different porcine BF-haplotypes. Address correspondence and offprint requests to: Luc J. Peelman.     

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.     

Microrecombinations generate sequence diversity in the murine major histocompatibility complex: analysis of the Kbm3, Kbm4, Kbm10, and Kbm11 mutants.

The mechanism that generates spontaneous mutants of the Kb histocompatibility gene was analyzed. Nucleotide sequence analysis of four mutant genes (Kbm3, Kbm4, Kbm10, and Kbm11) revealed that each mutant K gene contains clustered, multiple nucleotide substitutions. Hybridization analyses of parental B6 genomic DNA and cloned class I genes with mutant-specific oligonucleotide probes, followed by sequence analyses, have identified major histocompatibility complex class I genes in the K, D, and Tla regions (K1, Db, and T5, respectively) that contain the exact sequences as substituted into mutant Kb genes. These data provide evidence for the hypothesis that the mutant Kb genes are generated by a microrecombination (gene conversion) mechanism that results in the transfer of small DNA segments from class I genes of all four regions of the major histocompatibility complex (K, D, Qa, and Tla) to Kb. Many of the nucleotides substituted into the mutant Kb genes were identical to those found in other naturally occurring K alleles such as Kd. Thus, we propose that the accumulation of microrecombination products within the K genes of a mouse population is responsible for the high sequence diversity among H-2 alleles.