Molecular organization of the canine major histocompatibility complex

The major histocompatibility complex (MHC) is composed of a tightly linked cluster of genes; in dogs, this is referred to as the dog leukocyte antigen (DLA) region. The canine MHC is located on chromosome 12, and several genes within the DLA region have been identified that have significant sequence similarity to their human counterparts. However, in order to characterize other loci in the DLA region, DNA sequencing has begun using a canine bacterial artificial chromosome (BAC) library. Initially 135 BAC clones were isolated from a BAC library using a mixture of human and canine probes. These BAC clones were screened with locus-specific primers in polymerase chain reactions (PCRs). Fifty-six BAC clones were subjected to FingerPrinted Contig (FPC) analysis and several overlapping clones were identified. One BAC clone RP81-231-G24 has been sequenced. Preliminary sequence analysis of this 150 kb clone indicates that it contains the region where the class I and class III regions are joined and encompasses DLA-12a, DLA-53, DLA-12, DLA-64, TNF-alpha, and a canine gene that appears to resemble the HLA class III gene HSPA1A (HSP70-1).     

Odour signals major histocompatibility complex genotype in an Old World monkey

The major histocompatibility complex (MHC) is an extraordinarily diverse cluster of genes that play a key role in the immune system. MHC gene products are also found in various body secretions, leading to the suggestion that MHC genotypes are linked to unique individual odourtypes that animals use to assess the suitability of other individuals as potential mates or social partners. We investigated the relationship between chemical odour profiles and genotype in a large, naturally reproducing population of mandrills, using gas chromatography–mass spectrometry and MHC genotyping. Odour profiles were not linked to the possession of particular MHC supertypes. Sex influenced some measures of odour diversity and dominance rank influenced some measures of odour diversity in males, but not in females. Odour similarity was strongly related to similarity at the MHC, and, in some cases, to pedigree relatedness. Our results suggest that odour provides both a cue of individual genetic quality and information against which the receiver can compare its own genotype to assess genetic similarity. These findings provide a potential mechanism underlying mate choice for genetic diversity and MHC similarity as well as kin selection.     

Molecular variation of human major histocompatibility complex DQw3 beta-chains

Histocompatibility leukocyte antigen DQ molecules exhibit polymorphism of both DQ alpha- and beta-chains. Histocompatibility leukocyte antigen-DQw3 is associated with both DR4 and DR5 and can be further subdivided by reactivity with the monoclonal antibody TA10. To determine the molecular nature of the DQ polymorphic alleles associated with the DR4 haplotype, we have sequenced and analyzed DQ alpha and beta cDNA clones obtained from a DR4, Dw4, DQw3 cell line which is TA10-positive. The DQ alpha-chain sequence was identical to previously published sequences from the DR4 haplotype, but the DQ beta sequence differed from published DR4-DQ beta sequences obtained from DQw3-positive TA10-negative cell lines by eight amino acids, six of which were located in the beta 1 domain. Thus, the TA10 serologic determinants reside on the DQ beta-chain. A TA10-specific oligonucleotide probe was constructed based on the DQ beta sequence, and its specificity was confirmed in a panel of TA10-positive and TA10-negative cell lines. An additional band was observed in Southern blotting experiments which may indicate a donor sequence for gene conversion.     

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

We used PCR to test various protocols and define a technique for DNA extraction directly from chicken-shed stool samples for the identification of Eimeria species that parasitize birds. It was possible to extract and amplify DNA of seven Eimeria species from field stool samples, using both protocols tested; extractions made with phenol/chloroform protocols gave the best results. The primers were specific and sensitive, allowing amplification of samples containing as few as 20 oocysts, both in individual and in a multiplex PCR. Individualized PCR with the phenol/chloroform DNA extraction protocol detected a larger number of Eimeria species. Molecular diagnosis was found to be practical and precise, and can be used for monitoring and epidemiological studies of Eimeria.     

Systematic identification and characterization of chicken (Gallus gallus) ncRNAs

Recent studies have demonstrated that non-coding RNAs (ncRNAs) play important roles during development and evolution. Chicken, the first genome-sequenced non-mammalian amniote, possesses unique features for developmental and evolutionary studies. However, apart from microRNAs, information on chicken ncRNAs has mainly been obtained from computational predictions without experimental validation. In the present study, we performed a systematic identification of intermediate size ncRNAs (50–500 nt) by ncRNA library construction and identified 125 chicken ncRNAs. Importantly, through the bioinformatics and expression analysis, we found the chicken ncRNAs has several novel features: (i) comparative genomic analysis against 18 sequenced vertebrate genomes revealed that the majority of the newly identified ncRNA candidates is not conserved and most are potentially bird/chicken specific, suggesting that ncRNAs play roles in lineage/species specification during evolution. (ii) The expression pattern analysis of intronic snoRNAs and their host genes suggested the coordinated expression between snoRNAs and their host genes. (iii) Several spatio-temporal specific expression patterns suggest involvement of ncRNAs in tissue development. Together, these findings provide new clues for future functional study of ncRNAs during development and evolution.     

Molecular docking of superantigens with class II major histocompatibility complex proteins

The molecular recognition of two superantigens with class II major histocompatibility complex molecules was simulated by using protein– protein docking. Superantigens studied were staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) in their crystallographic assemblies with HLA-DR1. Rigid-body docking was performed sampling configurational space of the interfacial surfaces by employing a strategy of partitioning the contact regions on HLA-DR1 into separate molecular recognition units. Scoring of docked conformations was based on an electrostatic continuum model evaluated with the finite-difference Poisson– Boltzmann method. Estimates of nonpolar contributions were derived from the buried molecular surface areas. We found for both superantigens that docking the HLA-DR1 surface complementary with the SEB and TSST-1 contact regions containing a homologous hydrophobic surface loop provided sufficient recognition for the reconstitution of native-like conformers exhibiting the highest-scoring free energies. For the SEB complex, the calculations were successful in reproducing the total association free energy. A comparison of the free-energy determinants of the conserved hydrophobic contact residue indicates functional similarity between the two proteins for this interface. Though both superantigens share a common global association mode, differences in binding topology distinguish the conformational specificities underlying recognition. © 1998 John Wiley & Sons, Ltd.     

Biochemical identification of B-F and B-G allelic variants of the chicken major histocompatibility complex

Biochemical methods were used to analyse B-F and B-G antigens of the chicken major histocompatibility complex (MHC). In a panel of 12 inbred or partially inbred chicken lines the MHC haplotypes, originally defined by serological and histogenetical methods, were compared. Using monoclonal 18-6G2, allele-specific B-G patterns were obtained by immunoblotting. Comparison of B-G12 and B-G2 revealed a shared banding pattern, but additional products were detected for B-G12. The B-F products of B2 and B12 had identical IEF patterns. The identical B-F products and partially shared B-G products might explain the serological cross-reaction between these haplotypes. In addition, the IEF pattern of B-F21 appeared similar to B-F2 and B-F12, but the partial proteolysis map showed a clear difference. Although two B-F bands could be detected per haplotype, no evidence for the expression of more than one B-F locus was found. The biochemical methods enabled a precise definition of expressed MHC products and can be a useful tool for the identification of B-alleles in other chicken lines or outbred chickens for their MHC antigens.     

The major histocompatibility complex in the chicken

The chicken B complex is the first non-mammalian MHC characterized at the molecular level. It differs from the human HLA and murine H-2 complexes in the small size of the class I (B-F) and class II (B-L) genes and their close proximity. This proximity accounts for the absence of recombination between B-F and B-L genes and leaves no space for class III genes. Moreover the B-F and B-L genes are tightly linked to unrelated genes absent from mammalian MHCs, such as the polymorphic B-G genes and a member of the G protein beta subunit family. This linkage could form the basis for resistance to viral-induced tumors associated with some B complex haplotypes.     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

The major histocompatibility complex of primates

The major histocompatibility complex (MHC) encodes cell surface glycoproteins that function in self-nonself recognition and in allograft rejection. Among primates, the MHC has been well defined only in the human; in the chimpanzee and in two species of macaque monkeys the MHC is less well characterized. Serologic, biochemical and genetic evidence indicates that the basic organization of the MHC linkage group has been phylogenetically conserved. However, the number of genes and their linear relationship on the chromosomes differ between species. Class I MHC loci encode molecules that are the most polymorphic genes known. These molecules are ubiquitous in their tissue distribution and typically are recognized together with nominal antigens by cytotoxic lymphocytes. Class II MHC loci constitute a smaller family of serotypes serving as restricting elements for regulatory T lymphocytes. The distribution of class II antigens is limited mainly to cell types serving immune functions, and their expression is subject to up and down modulation. Class III loci code for components C2, C4 and Factor B (Bf) of the complement system.Interspecies differences in the extent of polymorphism occur, but the significance of this finding in relation to fitness and natural selection is unclear. Detailed information on the structure and regulation of MHC gene expression will be required to understand fully the biologic role of the MHC and the evolutionary relationships between species. Meanwhile, MHC testing has numerous applications to biomedical research, especially in preclinical tissue and organ transplantation studies, the study of disease mechanisms, parentage determination and breeding colony management. In this review, the current status of MHC definition in nonhuman primates will be summarized. Special emphasis is placed on the CyLA system of M. fascicularis which is a major focus in our laboratory. A highly polymorphic cynomolgus MHC has been partially characterized and consists of at least 14 A locus, 11 B locus, 7 C locus class I allelic specificities, 9 Ia-like class II antigens and 6 Bf (class III) variants.     

Parasite immunity and the major histocompatibility complex

Parasite infestations offer fertile ground for investigation of the relationship between immunity, disease and the major histocompatibility complex (MHC). However, due to the complexities of parasite life cycles and the success of parasites in evading the immune response, immune reactions against the parasite often do not parallel protective immunity, and immunity does not imply lack of disease. — An additional level of complexity is introduced in some forms of parasite immunity by accessory effector cells, e. g., macrophages and eosinophils, that need to be activated for maximal effectiveness, and the activated form of these cells may partly compensate for a deficiency in specific immune responses. — It is not surprising, therefore, that polygenic effects operate in parasite immunity and reports linking non-MHC genes with parasite immunity far out number those linking MHC genes with it. From the reports that do link MHC genes with parasite immunity, two areas emerge that are interesting. First, the increased incidence of certainHLA genes in people with schistosomiasis who develop hepatosplenic disease may pinpoint individuals at risk of morbidity and direct early treatment to them. Second, mechanisms that intimately involve MHC products but are not linked to a particular MHC haplotype, may indicate newer areas in the investigation of parasite immunity.     

Molecular analysis distinguishes two HLA-DR3-bearing major histocompatibility complex extended haplotypes

We detected restriction fragment length polymorphisms that distinguish the extended haplotype HLA-B8,DR3,SCO1 from HLA-B18,DR3,F1C30 at the DR beta and DQ beta loci with five of seven restriction endonucleases used. One set of restriction fragments was always found on HLA-B8,DR3,SCO1 and associated with DRw52a, while the other was present on HLA-B18, DR3,F1C30 and correlated with DRw52b (the gene encoding the subtype of DRw52 associated with the BO1 or LB-Q1 antigen). Furthermore, using a full-length DQ beta gene probe, we found division in the DQw2 haplotype, in which DQw2a always associated with HLA-B8, DR3,SCO1, while DQw2b always occurred with HLA-B18,DR3,F1C3O. Our evidence thus indicates that serologically defined HLA-DR3, HLA-DRw52, and HLA-DQw2 are each produced by two structurally very different sets of genes, one set occurring in HLA-B8, DR3,SCO1, and the other in HLA-B18,DR3,F1C30.Abbreviations used in this paper BSA bovine serum albumin - MHC major histocompatibility complex - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate