cDNA cloning and genetic polymorphism of the swine major histocompatibility complex (SLA) class II DMA gene

cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM alpha chain were isolated using the polymerase chain reaction (PCR) products from the third exon in the human HLA-DMA gene as a probe. Amino acid comparative analysis revealed that these clones were more closely related to the bovine and human DMA genes than to the other swine class II genes alpha chain genes, DRA, DQA and DOA. These results suggest that the SLA-DMA gene is expressed and may function, like HLA-DM, as an important modulator in class II restricted antigen processing in swine. Furthermore, based on the sequences and PCR-restriction fragment length polymorphism (PCR-RFLP) patterns in the SLA-DMA gene, no allelic variation was recognized in the second exon, but five allelic variations were recognized in the third exon in five different breeds of swine. These DMA alleles were defined by variation at four nucleotide positions. Two of these alleles resulted in an amino acid substitution. These results suggest that SLA-DMA has little polymorphism as observed in HLA-DMA and mouse H2-Ma.     

Gene organization of the quail major histocompatibility complex (MhcCoja) class I gene region

 Class I genomic clones of the quail (Coturnix japonica) major histocompatibility complex (MhcCoja) were isolated and characterized. Two clusters spanning the 90.8 kilobase (kb) and 78.2 kb class I gene regions were defined by overlapping cosmid clones and found to contain at least twelve class I loci. However, unlike in the chicken Mhc, no evidence for the existence of any Coja class II gene was obtained in these two clusters. Based on comparative analysis of the genomic sequences with those of the cDNA clones, Coja-A, Coja-B, Coja-C, and Coja-D (Shiina et al. 1999), these twelve loci were assigned to represent one Coja-A gene, two Coja-B genes (Coja-B1 and -B2), four Coja-C genes (Coja-C1-C4), four Coja-D genes (Coja-D1-D4), and one new Coja-E gene. A class I gene-rich segment of 24.6 kb in which five of these genes (Coja-B1, -B2, -D1, -D2 and -E) are densely packed were sequenced by the shotgun strategy. All of these five class I genes are very compact in size [2089 base pairs (bp)–2732 bp] and contain no apparent genetic defect for functional expression. A transporter associated with the antigen processing (TAP) gene was identified in this class I gene-rich segment. These results suggest that the quail class I region is physically separated from the class II region and characterized by a large number of the expressible class I loci (at least seven) in contrast to the chicken Mhc, where the class I and class II regions are not clearly differentiated and only at most three expressed class I loci so far have been recognized. Received: 9 March 1998 / Revised: 12 October 1998     

Isolation and characterization of cDNA clones for chicken major histocompatibility complex class II molecules

The chicken major histocompatibility complex (MHC), the B complex, is being intensively analysed at the DNA level. To further probe the molecular structure of chicken MHC class II genes, cDNA clones coding for chicken MHC class II (B-L) p chain molecules were isolated from an inbred G-B2 Leghorn chicken spleen and liver. Twenty-nine cDNA clones were isolated from the spleen and eight cDNA clones were isolated from the liver. Based on restriction maps, most clones could be clustered into one family of genes. Four cDNA clones were sequenced (S7, S10 and S19 from the spleen and L1, which was identical to S19, from the liver). Complete amino acid sequences of B-Lβ chain molecules were predicted from the nucleotide sequences of the cDNA clones. Although both the nature and the location of the conserved residues were similar in chicken and mammalian sequences, some species-specific differences were found, suggesting that the structures of the B-L molecules of this haplotype are similar, but not identical, to their mammalian counterparts.     

白假丝酵母激活的巨噬细胞自噬促进MHCⅡ抗原提呈

目的 探究白假丝酵母（Candida albicans）激活的Raw264.7细胞自噬在主要组织相容性复合体Ⅱ类分子（MHCⅡ）抗原提呈以及协同刺激分子表达中的作用。方法 C. albicans刺激Dectin-1单克隆抗体封闭或白皮杉醇阻断的Raw264.7细胞,Western blot法检测LC3Ⅱ表达量,RT-PCR检测CD80与CD86的表达。免疫荧光实验观察有无3-MA预处理的GFP-LC3-Raw264.7细胞与C. albicans共孵育后MHCⅡ与LC3在胞浆的分布,ELISA法检测有无封闭Dectin-1或3-MA预处理的Raw264.7细胞与C. albicans共孵育不同时间段后IL-6的分泌。结果 阻断Dectin-1或Sky后C. albicans诱导的LC3Ⅱ表达降低。LC3、MHCⅡ与胞内C. albicans存在显著的共定位关系,阻断自噬后C. albicans与MHCⅡ的共定位明显减弱。C. albicans引发Raw264.7细胞表达CD80与CD86 mRNA,封闭Dectin-1或阻断自噬后二者转录水平降低。C. albicans通过Dectin-1引发Raw264.7细胞分泌IL-6,阻断自噬对IL-6分泌无显著影响。结论 C. albicans通过Dectin-1/Sky通路激活巨噬细胞自噬,自噬体的构建促进MHCⅡ招募至胞内C. albicans,并促进协同刺激分子的表达。     

Expression and characterization of ovine major histocompatibility complex class II (OLA-DR) genes

Previous work made use of nucleic acid probes corresponding to different subtypes of the class II regions of the human and murine major histocompatibility complex (MHC) to isolate seven different alpha and 24 different beta genes of the ovine MHC from two cosmid libraries. In an attempt to identify pairs of alpha and beta genes capable of cell surface expression, all permutations of alpha and beta genes were in turn transfected into mouse L-cells. Two pairs of alpha and beta genes co-expressed and stable ovine MHC class II L-cell lines were developed. The expressed alpha genes had previously been defined as DR-alpha homologues (DRA) by differential Southern hybridization to human subtype specific class II probes. The expressed ovine beta genes were also assigned as ovine DR-beta homologues (DRB) on the basis of their sequence having a higher degree of similarity with human DRB than any other subtype. A total of eight out of 23 anti-sheep class II specific monoclonal antibodies were typed OLA-DR specific by FACScan analysis using the L-cell lines.     

Effect of invariant chain on major histocompatibility complex class I molecule expression and stability on human breast tumor cell lines

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.     

Class I and class II MHC bind self peptide sets that are strikingly different in their evolutionary characteristics

 Comparison of peptides eluted from human class I and class II major histocompatibility complex (MHC) molecules and the proteins from which they are derived (source proteins) revealed that class I MHC bind peptides derived from proteins that are highly conserved, hydrophilic, and universally expressed, while the peptides themselves are hydrophobic and even more conserved than their source proteins. In contrast, source proteins for class II-bound peptides were not significantly more conserved than a random sample of proteins. Class II-bound peptides were generally more conserved than their source proteins but were significantly less conserved than class I-bound peptides. The characteristics of class I-bound peptides can probably be explained by the selectivity of processing and transport of peptides for binding by class I, while the relative lack of selectivity of peptide binding for class II may explain the high incidence of autoimmune diseases associated with alleles of these molecules. Received: 17 May 1999 / Revised: 5 August 1999     

Sequence variation at the major histocompatibility complex DRB loci in beluga (Delphinapterus leucas) and narwhal (Monodon monoceros)

 The variation at loci with similarity to DRB class II major histocompatibility complex loci was assessed in 313 beluga collected from 13 sampling locations across North America, and 11 narwhal collected in the Canadian high Arctic. Variation was assessed by amplification of exon 2, which codes for the peptide binding region, via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Two DRB loci were identified in beluga: DRB1, a polymorphic locus, and, DRB2, a monomorphic locus. Eight alleles representing five distinct lineages (based on sequence similarity) were found at the beluga DRB1 locus. Although the relative number of alleles is low when compared with terrestrial mammals, the amino acid variation found among the lineages is moderate. At the DRB1 locus, the average number of nonsynonymous substitutions per site is greater than the average number of synonymous substitutions per site (0.0806 : 0.0207, respectively;P<0.01). Most of the 31 amino acid substitutions do not conserve the physiochemical properties of the residue, and 21 of these are located at positions implicated as forming pockets responsible for the selective binding of foreign peptide side chains. Only DRB1 variation was examined in 11 narwhal, revealing a low amount of variation. These data are consistent with an important role for the DRB1 locus in the cellular immune response of beluga. In addition, the ratio of nonsynonymous to synonymous substitutions is similar to that among primate alleles, arguing against a reduction in the balancing selection pressure in the marine environment. Two hypotheses may explain the modest amount of Mhc variation when compared with terrestrial mammals: small population sizes at speciation or a reduced neutral substitution rate in cetaceans. Received: 15 July 1997 / Revised: 24 March 1998     

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine Λgt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class IIA sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.     

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.     

Isolation and characterization of major histocompatibility complex (MHC) class II B genes in the Barn owl (Aves: Tyto alba)

We isolated major histocompatibility complex class II B (MHCIIB) genes in the Barn owl (Tyto alba). A PCR-based approach combined with primer walking on genomic and complementary DNA as well as Southern blot analyses revealed the presence of two MHCIIB genes, both being expressed in spleen, liver, and blood. Characteristic structural features of MHCIIB genes as well as their expression and high non-synonymous substitution rates in the region involved in antigen binding suggest that both genes are functional. MHC organization in the Barn owl is simple compared to passerine species that show multiple duplications, and resembles the minimal essential MHC of chicken.