New recombinants within the MHC (B-complex) of the chicken

In a search for genetic recombinations within the major histocompatibility complex (MHC) of the chicken, the B-complex, the offspring from matings between heterozygous B15/B21 and B4/B6 animals were analysed by red cell agglutination. Among the progeny, 8,912 informative typings were performed. Four recombinants were found, all separating the B-complex loci B-F and B-G (B-F codes for Class I antigens, B-G codes for an antigen of which there is no known homologue in mammals). B-L (Class II antigen) always followed B-F. Stimulation in graft versus host reactions and in mixed lymphocyte cultures followed B-F/B-L. The mapping distance between the two loci B-F and B-G is in the range of 0.04 centimorgan. The lack of recombinants separating individual B-F loci in this study and in the studies of others might indicate that chicken MHC is less complex than those of mammalian species, but alternative explanations are also possible. So far no serologically defined recombinant separating Class I (B-F) and Class II (B-L) loci has been found.     

Recombination within the human MHC

Summary: Recombination (crossing over) in the human MHC is thought to have played a role in generation of novel alleles at various HLA loci. It is also responsible for the diversity observed at the haplotype level, although the functional consequences of this activity are not clear. Historic and family studies of recombination have provided estimations of recombination fractions across the MHC and identified potential hotspots for recombination in the class II region. Other characteristics of recombination in the human MHC such as haplotype specificity in recombination frequency and localized sequence motifs involved in recombination have been considered, but have been difficult to address given the constraints of human population studies, Single-sperm typing holds promise in overcoming some of the limitations inherent in the study of recombination in human populations. Both family-based and sperm typing analyses of recombination, along with our knowledge of linkage disequilibrium patterns in the MHC, may provide novel information regarding the evolution of HLA haplotypes chat will be difficult to obtain by other means.     

Retrospective evidence that the MHC (B haplotype) of chickens influences genetic resistance to attenuated infectious bronchitis vaccine strains in chickens.

Infectious bronchitis is a respiratory disease of chickens that is caused by the coronavirus infectious bronchitis virus (IBV). Virtually all broiler and layer breeder flocks are routinely vaccinated against IBV. Two hatches of 1-day-old chicks from four lines were mistakenly vaccinated for infectious bronchitis using a moderately attenuated vaccine designed for chicks of an older age. The vaccination resulted in high mortality, and chicks from three of four lines died with signs typical of infectious bronchitis. The mortality that occurred using this less-attenuated vaccine was significantly influenced by the genetic line, and the MHC (B) haplotype in chickens of three B congenic lines. B congenic chickens possessing the B*15 haplotype were resistant in contrast to chickens possessing the B*13 or B*21 haplotypes. Chicks from two further hatches of the four lines were vaccinated appropriately with a more attenuated IBV vaccine, and only limited chick mortality was seen. These retrospective data from two repeated hatches confirm earlier data indicating chicken genes influence resistance to IBV, and indicate for the first time that genes tightly linked to the B haplotype are relevant in resistance to IBV. Due to extenuating circumstances it was not possible to verify results with chicks from F2 matings. Factors that may enhance definition of the role of the B haplotype in immune response to IBV, and the desirability for further analysis of a B haplotype-linked influence on immunity to IBV are discussed.     

B-complex genetic control of immune response to HSA, (T,G)-A--L, GT and other substances in chickens.

Immune response to poly-(L-tyrosine-L-glutamic acid)-poly-D,L-alanine-poly-L-lysine (T,G)-A--L), human serum albumin (HSA), and (L-glutamic acid50, L-tyrosine50)n (GT) was found to be linked to the B complex in an outbred line of Leghorns segregating for the B1, B2, and B19 alleles. Birds of the blood group genotypes B1B1, B2B2, and B19B19 were low, intermediate, and high responders, respectively to either (T,G)-A--L or HSA. Response to GT, however, differed, with the B2B2 genotype being the only responder. No real genotype differences in immune response to DNP-congugates and sheep red blood cells (SRBC) could be detected.     

Equine leucocyte antigen system. IV. Recombination within the major histocompatibility complex (MHC)

A case of recombination between the putative class I ELA antigen series and the structure(s) governing mixed lymphocyte reactivity in an informative horse family is described. The results of serological typing, 'lysostripping' and mixed lymphocyte culture tests strongly suggest that the recombination took place between two loci and is not intragenic. An alloantigenic membrane structure, provisionally called B1, which does not belong to the known ELA series, was also involved in the cross-over. The B1 antigen resembles the class II gene products of other species in two respects: it is not present on platelets, and alloantiserum with specificity for B1 inhibits the stimulatory effect of B1-carrying cells in mixed lymphocyte cultures. The B1 antigen does not follow the classical distribution, however, being expressed on both B and T lymphocytes. The finding of separate loci for the first series of ELA antigens and the MLR governing structure(s) demonstrates the similarity of the genetic organization of the horse MHC to that in other species.     

Hypomethylation is necessary but not sufficient for V(D)J recombination within a transgenic substrate

Although an inverse correlation between CpG methylation and V(D)J recombination has been demonstrated for both artificial substrates and endogenous genes, it is not known whether all hypomethylated targets are competent to rearrange or if other factors are required. We have created several artificial V(D)J recombination substrate transgenes whose methylation can be controlled by breeding into different genetic backgrounds. A transgene which contains the immunoglobulin heavy chain intronic enhancer rearranges efficiently in B lymphocytes when the transgene loci are unmethylated. When the same loci become methylated, upon breeding into a different mouse strain, no rearrangement can be detected. A similar transgene, but lacking the enhancer, also shows no evidence of V(D)J recombination when it is methylated. Even when this enhancerless transgene is hypomethylated, however, no V(D)J recombination can be detected in B lymphocytes. Thus, hypomethylation is required to permit V(D)J recombination but not all hypomethylated targets are capable of recombination. The results may indicate that the immunoglobulin enhancer is required for the assembly of factors involved in V(D)J recombination.     

Infectious bronchitis virus: evidence for recombination within the Massachusetts serotype

The spike (S) glycoprotein gene (which encodes two subunits, S1 and S2), the membrane (M) glycoprotein gene and the gene which encodes the products of gene 3 are situated in the infectious bronchitis virus (IBV) genome in the order S1-S2-3-M. The S1 gene of four isolates of the Massachusetts (Mass) serotype, isolated between 1970 and 1984, each differed by only 2 to 3% from that of older Mass serotype isolates, including M41 and H120. Similarly, sequencing of the end of gene 3 and the beginning of the M gene showed that three of the isolates differed from the older Mass isolates by 2% or less. In contrast, the fourth Mass strain, Portugal/322/82, differed by 11% and 24% in gene 3, and the first part of gene M, respectively, suggesting that this strain was a recombinant. It was not possible to identify a putative recombination site but the finding that the S2 gene of Portugal/322/82 differed from other strains much less than did the S1 gene suggests that recombination might have occurred in S2 as a consequence of the high S2 nucleotide homology among IBV strains.     

Assembly of MHC class I molecules within the endoplasmic reticulum

MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.     

The polymerases for V(D)J recombination

DNA transactions of a wide variety generally require three major types of enzymatic activities: nucleases, polymerases, and ligases. V(D)J recombination is no exception. In this issue, Bertocci et al. (2006) have provided new insight by generating mice deficient in one or more of the polymerases.     

Genomic characterization of novel dolphin papillomaviruses provides indications for recombination within the Papillomaviridae

Phylogenetic analysis of novel dolphin (Tursiops truncatus) papillomavirus sequences, TtPV1, -2, and -3, indicates that the early and late protein coding regions of their genomes differ in evolutionary history. Sliding window bootscan analysis showed a significant a change in phylogenetic clustering, in which the grouped sequences of TtPV1 and -3 move from a cluster with the Phocoena spinipinnis PsPV1 in the early region to a cluster with TtPV2 in the late region. This provides indications for a possible recombination event near the end of E2/beginning of L2. A second possible recombination site could be located near the end of L1, in the upstream regulatory region. Selection analysis by using maximum likelihood models of codon substitutions ruled out the possibility of intense selective pressure, acting asymmetrically on the viral genomes, as an alternative explanation for the observed difference in evolutionary history between the early and late genomic regions of these cetacean papillomaviruses.     

Identification of independent risk loci for Graves' disease within the MHC in the Japanese population

To identify genetic variants that confer the risk of Graves' disease (GD) in the Japanese population, we conducted a two-stage genome-wide association study (GWAS) using 1119 Japanese individuals with GD and 2718 unrelated controls, and a subsequent replication study using independent 432 GD cases and 1157 controls. We identified 34 single nucleotide polymorphisms (SNPs) to be significantly associated with GD in the GWAS phase. Twenty-two out of 34 SNPs remained positive in the replication study. All 22 SNPs were located within the major histocompatibility complex (MHC) locus on chromosome 6p21. No strong long-range linkage disequilibrium (LD) was observed among the 22 SNPs, indicating independent involvement of multiple loci within the MHC with the risk of GD. Multivariate stepwise logistic regression analysis selected rs3893464, rs4313034, rs3132613, rs4248154, rs2273017, rs9394159 and rs4713693, as markers for independent risk loci for GD. The analysis of LD between these seven SNPs and tagging SNPs for GD-associated human leukocyte antigen (HLA) alleles in the Japanese population (HLA-DPB1(*)0501 and HLA-A(*)0206) demonstrated that all of and five of seven SNPs were not in strong LD with HLA-DPB1(*)0501 and HLA-A(*)0206, respectively. Although causal variants remain to be identified, our results demonstrate the existence of multiple GD susceptibility loci within the MHC region.     

视网膜母细胞瘤基因内VNTR的多态分析

VNTR区是连锁分析和个体鉴别的有效遗传标志。本文采用PCR方法扩增了人类RB1基因第20内含子的VNTR多态位点,扩增产物经6％聚丙烯酰胺凝胶电泳,银染显示。结果表明该多态位点由多个等位片段构成,片段的大小在300－400bp之间。在108例无亲缘关系的个体中,我们检测到76例为杂合子,杂合子频率为70％。此外,我们还探讨了RB1．20VNTR在视网膜母细胞瘤产前诊断中应用的可能性。     

Cellular expression of MHC glycoproteins on erythrocytes from normal and aneuploid chickens

The major histocompatibility complex (MHC) in the chicken (B-complex) encodes glycoproteins homologous in function and distribution to the mammalian MHC. These are the B-F (class I) and B-L (class II) glycoproteins. In addition, a third glycoprotein (B-G) is also encoded by the chicken MHC. We are interested in examining gene regulation and cellular expression of these MHC gene products in the chicken. The trisomic line of chickens is being developed as an animal model for this purpose. Birds from this line contain either 2, 3, or 4 MHC-encoding chromosomes. In this study, we investigated the hypothesis that the quantities of B-complex glycoproteins on the membranes of fully differentiated erythrocytes are proportional to the number of MHC-encoding chromosomes present in particular birds. Hemagglutination final titer and quantitative adsorption assays were carried out using erythrocytes from disomic and aneuploid chickens homozygous for the B15 haplotype. The average hemagglutination final titers were higher for tetrasomic cells as compared to disomic cells. Furthermore, in adsorption assays, employing a B15 cross-reacting alloantiserum, trisomic and tetrasomic erythrocytes displayed increased adsorption capabilities (1.6 and 3.1 fold, respectively) compared to disomic control cells. These results indicate a step-wise increase in the amounts of erythrocyte surface glycoproteins per cell in the trisomics and tetrasomics, respectively. Such findings are consistent with a MHC-dosage-dependent model of gene expression in homeothermic vertebrates.     

Evolution of the mammalian MHC: natural selection, recombination, and convergent evolution

Summary: The genes that encode molecules involved in antigen presentation within the class I and class II regions of the mammalian major histocompatibility complex (MHC) include several that are highly polymorphic. There is evidence that this polymorphism is maintained by positive selection, most likely overdominant selection, relating to their role in presenting foreign peptides to T cells. This selection can maintain allelic lineages for much longer periods of time than neutral polymorphisms are expected to last, but sharing of polymorphic amino acid motifs among species of different mammalian orders is due to independent (or convergent) evolution rather than common ancestry. It has been suggested that interallelic recombination (gene conversion) plays a role in enhancing polymorphism, but there is evidence of striking differences among loci with respect to the rate at which such recombination has contributed to current polymorphism. Recent attempts to interpret linkage relationships in the MHC region as evidence of ancient genomic duplications are not supported by phylogenetic analysis. Rather, natural selection may have played a role in the linkage of other genes to those of the MHC.     

Identification of the recombination site within the steroid 21-hydroxylase gene (CYP21) of the HLA-B47,DR7 haplotype.

The HLA haplotype A3-Cw6-B47-C4A91-BQ0-DR7 is associated with congenital adrenal hyperplasia (CAH), since it only carries the dysfunctional steroid 21-hydroxylase A pseudogene as well as the 5' adjacent complement C4A gene. The recombination site leading to the deletion of the complement C4B and steroid 21-hydroxylase B genes in this haplotype was studied by determining the 21-hydroxylase genomic DNA sequence in comparison to the standard CYP21A- and CYP21B-specific sequences. A 200-bp region between exons 7 and 8 was identified as a possible recombination site. Thus the deleted area comprises the 3' end of the CYP21A pseudogene, the entire C4B gene and the 5' end of the CYP21B gene. The findings were confirmed by PCR amplification of a 1.8-kb fragment of the CYP21 gene. This PCR system is specific for CYP21A/B recombinant genes and may be used for screening among CAH patients carrying this type of deletion.     

Peptide register shifting within the MHC groove: theory becomes reality

Degeneracy in immune recognition is usually thought of in terms of the astonishing ability of the T cell receptor to recognize an enormously diverse array of peptides bound to major histocompatibility complex (MHC) molecules. However, in this essay we discuss an alternative aspect of degeneracy in T cell recognition: the notion that peptides can assume different "registers" in the groove of a single MHC molecule, as first suggested and demonstrated by Sercarz and co-workers (reviewed in [J. autoimmun. 16 (2001) 201]). There is now abundant evidence, derived from functional, biochemical and structural studies, that single peptides can assume alternative, unpredictable binding registers by frameshifting within the MHC groove [Nat. Immunol. 3 (2002) 175;; J. Exp. Med. 187 (1998) 1505; J. Mol. Biol. 304 (2000) 177; Biochemistry 38 (1999) 16663; J. Exp. Med. 197 (2003) 1391; Eur. J. Immunol. 19 (1989) 681]. Hence, register shifting adds an additional dimension to the concept of degeneracy. In fact, the possibility of register shifting multiplies the universe of peptide-MHC (pMHC) surfaces that a TCR must recognize by an unknown, perhaps enormous factor. Register shifting also has profound implication for autoimmunity: (1) as a mechanism to "mask" autoantigenic epitopes during thymic education [Immunol. Rev. 169 (1999) 147; Immunity 17 (2002) 83]; and (2) as a possible source for pMHC complexes capable of molecular mimicry.     

Regulation of V(D)J recombination

Adaptive immunity is intimately linked to the expression of antigen-specific immunoglobulin and T cell receptor genes and their recombination assembly from germline V, D and J gene segments. This developmentally regulated process relies on the activity of the Rag1-Rag2 recombinase, on accessibility of target gene segments and on monoallelic gene activation. Recent studies have revealed new mechanisms that, along with recombinase activity and locus accessibility, are likely to contribute to the control of V(D)J recombination, including target-site bias by the recombinase, RNA processing and chromosome positioning.     

PAIRing for distal Igh locus V(D)J recombination

In this issue of Immunity Ebert et al. (2011) defined the lineage- and stage-specific Pax5-dependent cis-sequences termed PAIR elements in the distal region of the mouse heavy chain immunoglobulin locus (Igh). These sequences may have a role in long-range IgH V(D)J recombination.