Isolation and characterization of class I MHC genes in the giant panda (Ailuropoda melanoleuca)

Artificial breeding is an important project to protect,recover and reintroduce endangered species.Knowledge of the population’s genetic diversity at functional loci is important for the establishment of effective captive breeding programs.The major histocompatibility complex(MHC) genes are ideal candidate genetic markers to inform planned breeding,due to their high levels of polymorphism and importance in the main immune coding region of the vertebrate genome.In this study,we constructed BAC-based contigs and isolated six functional MHC class Ⅰ genes from the giant panda(Ailuropoda melanoleuca),which we designated Aime-C,Aime-F,Aime-I,Aime-K,Aime-L and Aime-1906.Analyses of the tissue expression patterns and full-length cDNA sequences of these class I genes revealed that Aime-C,-F,-I and-L could be considered classical class Ⅰ loci,due to their extensive expression patterns and normal exonic structures.In contrast,Aime-K and-1906 appeared to be nonclassical genes based on their tissue-specific expression patterns and the presence of an abnormal exon 7 in both genes.We established techniques for genotyping exons 2 and 3 of the classical loci using locus-specific single strand conformation polymorphism(SSCP) and sequence analysis.In the Chengdu captive population,we identified one monomorphic locus(Aime-F) and three polymorphic loci with different numbers of alleles(4/4/4 exon 2 alleles at Aime-C/I/L and 6/5/5 exon 3 alleles at Aime-C/I/L).The distributions of the Aime-C,-I and-L alleles among members of different families were in good agreement with the known pedigree relationships,suggesting that the genotyping results are reliable.Therefore,the MHC-I genotyping techniques established in this study may provide a powerful tool for the future design of scientific breeding or release/reintroduction programs.     

Antigen presenting function of class II MHC expressing pancreatic beta cells

Class II major histocompatibility complex (MHC) gene expression in the mouse is generally limited to thymic epithelium and bone marrow-derived cells such as B lymphocytes and cells of the macrophage/dendritic cell lineage (M phi/DC). Class II-bearing B lymphocytes and M phi/DC possess antigen presenting cell (APC) function; that is, they can stimulate T lymphocytes reactive to either antigen plus MHC or foreign MHC alone. To assess whether non-bone-marrow-derived cells can acquire APC function and elicit graft rejection through expression of class II, we studied transgenic pancreatic islet beta cells that express a foreign class II (I-E) molecule. In vivo, grafts of I-E+ transgenic islets into I-E- naive hosts are not rejected unless the host is primed by an injection of I-E+ spleen cells. In vitro, the I-E+ beta cells are unable to stimulate T lymphocytes reactive to I-E plus a peptide antigen. Paradoxically, they induce antigen specific unresponsiveness in the T cells. We propose that expression of class II on non-lymphoid cells may serve as an extrathymic mechanism for maintaining self tolerance.     

Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A

The major histocompatibility complex (MHC) on chromosome 6 is associated with susceptibility to more common diseases than any other region of the human genome, including almost all disorders classified as autoimmune. In type 1 diabetes the major genetic susceptibility determinants have been mapped to the MHC class II genes HLA-DQB1 and HLA-DRB1 (refs 1-3), but these genes cannot completely explain the association between type 1 diabetes and the MHC region. Owing to the region's extreme gene density, the multiplicity of disease-associated alleles, strong associations between alleles, limited genotyping capability, and inadequate statistical approaches and sample sizes, which, and how many, loci within the MHC determine susceptibility remains unclear. Here, in several large type 1 diabetes data sets, we analyse a combined total of 1,729 polymorphisms, and apply statistical methods-recursive partitioning and regression-to pinpoint disease susceptibility to the MHC class I genes HLA-B and HLA-A (risk ratios >1.5; P(combined) = 2.01 x 10(-19) and 2.35 x 10(-13), respectively) in addition to the established associations of the MHC class II genes. Other loci with smaller and/or rarer effects might also be involved, but to find these, future searches must take into account both the HLA class II and class I genes and use even larger samples. Taken together with previous studies, we conclude that MHC-class-I-mediated events, principally involving HLA-B*39, contribute to the aetiology of type 1 diabetes.     

Characterization of major histocompatibility complex DRA and DRB genes of the forest musk deer (Moschus berezovskii)

The forest musk deer(Moschus berezovskii) is one of the most endangered species in China.Over the past decades,extensive hunting and poaching have pushed the forest musk deer to the edge of extinction,and conservation biologists are presently pursuing scientific management plans to rescue this species.The major histocompatibility complex(MHC),a cluster of genes responsible for antigen presentation,is a highly polymorphic genomic region in vertebrates that has become a popular functional marker system for studying adaptive variation.In this study,we developed locus-specific genotyping primers for exon 2 fragments of one DRA gene and one DRB locus of the forest musk deer using a suite of comprehensive methods that included universal primer amplification,genome walking,single-strand conformation polymorphism(SSCP),heteroduplex(HD) profiling,and sequence analysis.Each forest musk deer showed no more than two sequences per locus,confirming the specificity of our primers.Genotyping with these primers allowed us to identify two DRA alleles and six DRB alleles in a captive breeding population of the Sichuan Musk Deer Breeding Institution.For the DRA locus,we found a slightly higher observed heterozygosity(N O =0.154) than expected(N E =0.143).In contrast,the DRB locus showed a significant heterozygote deficiency(N O =0.508;N E =0.761;P<0.05),which was probably due to inbreeding in the captive population.An obvious excess of nonsynonymous substitutions over synonymous was observed at the antigen-binding positions of the DRA and DRB loci,showing the presence of positive selection in the forest musk deer DR genes.Finally,generation of phylogenetic trees for the DRA and DRB sequences of the forest musk deer and other ruminants revealed that the DRA and DRB loci identified in this study had homologous relationships with the known ruminant DRA and DRB genes.Based on this analysis,and to facilitate future studies,we named these novel loci Mobe-DRA and Mobe-DRB3.     

The origin of MHC class II gene polymorphism within the genus Mus

The I region of the major histocompatibility complex (MHC) of the mouse (H-2) contains a tightly-linked cluster of highly polymorphic genes (class II MHC genes) which control immune responsiveness. Speculation on the origin of this polymorphism, which is believed to be essential for the function of the class II proteins in immune responses to disease, has given rise to two hypotheses. The first is that hypermutational mechanisms (gene conversion or segmental exchange) promote the rapid generation of diversity in MHC genes. The alternative is that polymorphism has arisen from the steady accumulation of mutations over long evolutionary periods, and multiple specific alleles have survived speciation (trans-species evolution). We have looked for evidence of 'segmental exchange' and/or 'trans-species evolution' in the class II genes of the genus Mus by molecular genetic analysis of I-A beta alleles. The results indicate that greater than 90% (28 out of 31) of the alleles examined can be organized into two evolutionary groups both on the basis of restriction site polymorphisms and by the presence or absence of a short interspersed nucleotide element (SINE). Using this SINE sequence as an evolutionary tag, we demonstrate that I-A beta alleles in these two evolutionary groups diverged at least three million years ago and have survived the speciation events leading to several modern Mus species. Nucleotide sequence comparisons of eight Mus m. domesticus I-A beta alleles representing all three evolutionary groups indicate that most of the divergence in exon sequences is due to the steady accumulation of mutations that are maintained independently in the different alleles. But segmental exchanges between alleles from different evolutionary groups have also played a role in the diversification of beta 1 exons.     

MiR1511 co-regulates with miR1511* to cleave the GmRPL4a gene in soybean

MicroRNA1511 (miR1511) is a small RNA with unknown function identified in several plants by deep sequencing. In this study, we showed that this small RNA is an authentic miRNA by analyzing the structure of the precursor stem-loop containing the newly identified miR1511* sequence. We confirmed this result by Northern blotting analysis. We used 5??RACE to identify one of the target genes (GmRPL4a) cleaved by both miR1511 and miR1511*. The site cleaved by miR1511* was located in the first exon of GmRPL4a, and the site cleaved by miR1511 was located in the second exon. The expression level of miR1511/1511* was higher in leaves than in roots and stems. In contrast, the lowest level of GmRPL4a expression was in the leaves and the highest in the root. These results indicate that an miRNA can co-regulate with an miRNA* to cleave the same target gene in plants, and that the level of GmRPL4a mRNA is regulated by miR1511/1511*.     

Characterization of MHC class II A genes in Hainan Eld’s deer (Cervus eldi hainanus)

The major histocompatibility complex(MHC) genes play pivotal roles in the immune system of vertebrates against antigens.They are also significant indicators of genetic structure,and are vital to species-level population viability analyses and disease risk assessments.In this study,two DRA and two DQA sequences were isolated from Hainan Eld’s deer(Cervus eldi hainanus) using rapid amplification of cDNA ends(RACE) and single-strand conformation polymorphism-heteroduplex(SSCP-HD) analysis.Nucleotide sequence analysis revealed large differences between the two DQA sequences,especially in their exon 2 regions,but only minimal differences between the variants of the DRA gene.Comparison of the predicted amino acid sequences of the Ceel-MHC class Ⅱ A variants with those from six other species revealed that these molecules share high homology among ruminants.A phylogenetic tree of four class Ⅱ A sequences from Hainan Eld’s deer and the other species placed the newly identified DQA and DRA genes on two distinct branches(100%-supportively),and further divided the two DQA sequences into 98%-supportive DQA1 and 99%-supportive DQA2 clusters,respectively.Therefore,this study identified monomorphic Ceel-DQA1 and Ceel-DQA2 genes,and one dimorphic Ceel-DRA gene from Hainan Eld’s deer.     

An amino-acid substitution involved in phenylketonuria is in linkage disequilibrium with DNA haplotype 2

Phenylketonuria (PKU) is an autosomal recessive human genetic disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase, EC 1.14.16.1). PKU is a common inborn error of amino-acid metabolism in caucasian populations and approximately 1 in 50 individuals are carriers of a PKU allele. To define the molecular basis of PKU, we characterized twelve restriction fragment-length polymorphism (RFLP) haplotypes of the PAH locus in the northern European population and observed that 90% of the PKU alleles in this population are confined to four common RFLP haplotypes. We have recently reported a splicing mutation in the PAH gene that is associated with RFLP haplotype 3 which is present at about 40% of mutant alleles. We now report the molecular lesion associated with the RFLP haplotype 2 mutant allele. This defect is caused by a C-to-T transition in exon 12 resulting in an amino-acid substitution (Arg to Trp) at residue 408 of PAH. Direct hybridization analysis of the point mutation using a specific oligonucleotide probe demonstrated that this mutation is also in linkage disequilibrium with RFLP haplotype 2 alleles that make up about 20% of mutant PAH genes.     

中国恒河猴MHC Ⅰ型部分等位基因的分析

目的对中国恒河猴主要组织相容性复合体（MHC）I型部分基因进行携带情况调查与分析。方法采用序列特异性引物（PCR-SSP）分型方法对华南灵长类动物研究中心繁殖的30只谱系清晰的中国恒河猴（Macacamulatta）的32个MHC I型分子位点进行检测。结果采用的32对引物中,中国恒河猴可检出携带23个MHC I等位基因,但基因携带频率存在很大的差异,由3.57%至82.14%不等。结合遗传谱系分析,判断A1＊21和A2＊05之间以及B＊04和B＊30之间可能就是连锁的。结论中国恒河猴携带能控制病毒复制的MHC I型基因位点的频率较高,其基因携带频率与已发表的印度恒河猴携带频率存在明显差异。本研究为促进中国恒河猴在AIDS研究中的应用,以及为建立携带特定MHC I基因实验猴小种群提供了依据。     

HLA-A and B polymorphisms predate the divergence of humans and chimpanzees

Major histocompatibility complex (MHC) glycoproteins bind processed fragments of proteins and present them to the receptors of T lymphocytes. The extraordinary polymorphism of class I MHC molecules in man (HLA-A, B and C) and mouse (H-2 K, D and L) poses many questions concerning their diversification and evolution. Comparison of allelic sequences within a species suggests diversity is generated by the assortment of point mutations into varied combinations by mechanisms of recombination and gene conversion. We have now compared class I MHC alleles in two closely related species: humans (Homo sapiens) and chimpanzees (Pan troglodytes). Chimpanzee homologues of HLA-A, HLA-B and a non-classical gene have been identified. No features distinguishing human and chimpanzee alleles could be found. Individual HLA-A or B alleles are more closely related to individual chimpanzee alleles than to other HLA-A or B alleles. These results show that a considerable proportion of contemporary HLA-A and B polymorphism existed before divergence of the chimpanzee and human lines. The stability of the polymorphism indicates that hyper-mutational mechanisms are not necessary to account for HLA-A, B and C diversity.     

HLA DRB1＊1501和DQB1＊0602与新疆维吾尔、汉族HPV感染及宫颈癌发生的相关性

探讨HLADRB1＊1501和DQB1＊0602与新疆维吾尔、汉族妇女HPV感染及宫颈癌发生的相关性。PCR-SSP和PCR检测287例浸润性宫颈癌（维族192例,汉族95例）及297例正常宫颈组织（维族203例,汉族94例）中DRB1＊1501和HLADQB1＊0602的分布频率和HPV16DNA。维族HPV16阳性NILM组中DRB1＊1501基因频率高于HPV16阴性NILM组（OR,2．222;95％CI,1．107—4．461;P=0．023）,差异有统计学意义;在维族ISCC组及HPV16阳性ISCC组中DQB1＊0602基因频率均低于对照组（OR,0．484;95％CI,0．324～0．722;P=0．000;OR,0．552;95％CI,0．360～0．845;P=0．006）,差异有统计学意义;汉族ISCC组中DRBl。1501等位基因及DRB1＊1501～DQB1＊0602单体型均低于对照组（分别为OR,0．305;95％CI,0．115～0．813;P=0．013和OR,0．274;95％CI,0．086—0．874;P=0．021）,差异有统计学意义。携带DRB1＊1501等位基因为新疆维族妇女HPV16易感基因。DQB1＊0602基因可能是新疆维族妇女宫颈癌的保护基因,而DRB1＊1501基因及DRB1＊1501～DQB1＊0602单体型则可能是新疆汉族妇女宫颈癌的保护基因。     

Signalling through the MHC class II cytoplasmic domain is required for antigen presentation and induces B7 expression.

Class II major histocompatibility complex (MHC) molecules function as antigen-presenting elements as well as signal transducers on B lymphocytes. We previously reported that a B lymphoma cell transfectant, 5C2, expressing genetically engineered I-Ak molecules with truncated cytoplasmic domains was severely impaired in both antigen presentation and in anti-Ia-induced intracytoplasmic signalling. These two functions could be restored by preculturing 5C2 cells with cyclic AMP analogues. Here we demonstrate that impaired signal transduction by truncated class II molecules results in a deficiency in induction of the newly defined B-cell accessory molecule B7 (ref. 8), which can be reversed by restoration of B7 expression. These data imply that contact of the T-cell antigen receptor with MHC/antigen ligand results in signal transmission through the class II cytoplasmic domain. This signal, which can be mimicked by dibutyryl cAMP, induces expression of B7, resulting in effective antigen presentation. The fact that crosslinking of surface class II MHC also induces B7 expression on normal resting human B cells supports this contention.     

Phosphatidylglycerol effect on oxygen-evolving activity in Ca2+-depleted photosystem II

The effect of anionic phosphatidylglycerol (PG) on oxygen evolution in a photosystem II (PS II ) particle depleted of Ca²⁺ (designated d_CaPSII ) has been investigated. The major finding is the observation of a new role of PG in the PSII function. That is, PG restores nearly the lost oxygen evolution in d_caPS II particle owing to Ca²⁺ depletion to the levels in intact PS II. Furthermore, there is a stimulation of oxygen-evolving activity in the d_CaPSII complexed with PG in the presence of exogenous CaCl₂, which PG enhances increasingly oxygen evolution with increasing CaCl₂ concentration. It is suggested that PG-induced oxygen evolution recovery of d_Ca PS II particle results from resumption of normal structure in protein by PG effect, whereas the enhancement of oxygen evolution in complex subject to CaCl₂ is ascribed to the optimization of such a structure due to coordination complex formation of Ca²⁺ ions with PG.     

Acetone hydrogenation in exothermic reactor of an isopropanol–acetone–hydrogen chemical heat pump: effect of intra-particle mass and heat transfer

Intra-particle mass and heat transfer plays an important role in performance of the exothermic fixed-bed reactor for an isopropanol-acetone-hydrogen chemical heat pump. In this work, an exothermic fixed-bed reactor model, taking into account the actual packing structure, is established in the commercial software Fluent. A 120° segment of a tube with tube-to-particle diameter ratio （n） of 4, where realistic particles are packed and set to porous media, is used to simulate the 3D external flow, concen- tration and temperature fields in the exothermic packed-bed reactor. The influence of catalyst particle diameter （dp） and micropore diameter （do） on the intra-particle temperature, species distribution, reaction rate and selectivity is dis- cussed. The appropriate dp and do are obtained. Simulation results showed that intra-particle temperature gradient is not obvious. Large dp and small do lead to remarkable gradient of reaction rate inside the catalyst particle and the decrease in the catalyst efficiency and reduce the acetone conversion and the selectivity in isopropanol. The optimal results reveal that the spherical catalyst with dp of 1 mm and dpore of 10 nm is appropriate for high-temperature acetone hydrogenation.     

A potential donor gene for the bm1 gene conversion event in the C57BL mouse

The mammalian major histocompatibility complex (MHC; H-2 complex in mouse) is a large multigene complex which encodes cell-surface antigens involved in the cellular immune response to foreign antigens. Class I polypeptides expressed at the H-2K and H-2D loci of numerous mouse strains exhibit an unusually high degree of genetic polymorphism, which is assumed to be related to their function as primary recognition elements in the immune response. We suggested that this H-2 polymorphism may arise by gene conversion-like events between non-allelic class I genes. This is supported by our recent comparison of the DNA sequences of the normal H-2Kb gene sequence, from the C57BL/10 mouse, and a mutant form of this gene called H-2Kbm1: the mutant allele differs from the H-2Kb gene in seven bases out of a region of 13 bases in exon 3 of the class I gene (which encodes alpha 2 (C1) the second highly polymorphic protein domain), suggesting that this region of new sequence had been introduced into the H-2Kb sequence following unequal pairing of two class I genes in the genome of the C57BL mouse. Schulze et al. have obtained similar results. Here we report work identifying a potential donor gene in our library of 26 class I genes cloned from the C57BL/10 mouse.     

Cycling of cell-surface MHC glycoproteins through primaquine-sensitive intracellular compartments

Class II major histocompatibility complex (MHC) glycoproteins associate with peptides derived from material endocytosed by antigen-presenting cells and processed along the endocytotic pathway. No consensus exists as to what extent class II molecules themselves are endocytosed and it is not known whether endocytosed MHC class II molecules can be recycled again to the cell surface--an itinerary which might allow a single cell-surface MHC molecule to associate with different peptides during its lifetime. We now show by using new cleavable labelling reagents that class II and class I MHC on B lymphoblastoid cells are continually endocytosed and recycled to the cell surface. The intracellular pool size is normally kept small by efficient recycling, but in the presence of primaquine the rate of recycling is slowed, thereby increasing the size of this pool substantially. On removal of the amine, the intracellular population recycles rapidly to restore the original distribution. These results reveal a cycle that might explain the rapid binding and turnover of some peptide/class II MHC complexes and the exchange of pre-existing for new peptides observed in living cells.     

First-principle study on the surface atomic relaxation properties of sphalerite

The surface properties of sphalerite (ZnS) were theoretically investigated using first principle calculations based on the density functional theory (DFT). DFT results indicate that both the (110) and the (220) surfaces of sphalerite undergo surface atom relaxation after geometry optimization, which results in a considerable distortion of the surface region. In the normal direction, i.e., perpendicular to the surface, S atoms in the first surface layer move outward from the bulk (d₁), whereas Zn atoms move toward the bulk (d₂), forming an S-enriched surface. The values of these displacements are 0.003 nm for d₁ and 0.021 nm for d₂ on the (110) surface, and 0.002 nm for d₁ and 0.011 nm for d₂ on the (220) surface. Such a relaxation process is visually interpreted through the qualitative analysis of molecular mechanics. X-ray photoelectron spectroscopic (XPS) analysis provides the evidence for the S-enriched surface. A polysulphide (S_n2-) surface layer with a binding energy of 163.21 eV is formed on the surface of sphalerite after its grinding under ambient atmosphere. This S-enriched surface and the S_n2- surface layer have important influence on the flotation properties of sphalerite.     

Molecular evolution of rice S 5 n and functional comparison among different sequences

The wide compatibility gene, S ₅ ⁿ , can overcome embryo sac sterility between indica and japonica subspecies of rice. Therefore, it is very important to characterize the features of the S ₅ ⁿ sequence to reveal the origin and evolution of S ₅ ⁿ . In this paper, 26 cultivated rice haplotypes and 22 wild rice accessions harboring S ₅ ⁿ were used to sequence S ₅ ⁿ . The results showed that 15 genotypes among the 48 materials were fully consistent with control cultivar 02428 (CK). The other 33 accessions had different degrees of variation in the S ₅ ⁿ sequence. Variations in the coding region mainly occurred in the second exon and eight materials showed a 10-bp deletion at 1710?C1719 bp, including wild (O. nivara) and cultivated rice, such as IRW501 and Yuetai B. S ₅ ⁿ sequences were not biased and evolved neutrally. The 48 materials could be divided into 4 categories using a phylogenetic tree of the amino acid sequences. Most of the wild rice clustered together, and the cultivated rice clustered into another group. Eight cultivated rice and O. nivara (wild rice) clustered in another group, which were found to lack 10 consecutive bases in exon 2. Eight rice varieties with high numbers of differences in their S ₅ ⁿ coding regions were crossed with testers (typically indica and japonica) to produced test cross F₁ populations. The F₁s were examined for their ability to overcome indica-japonica hybrid sterility. The result showed that the embryo sac fertility of S ₅ ⁿ -containing hybrids increased significantly compared with control hybrids, but there were no differences among the materials with divergent sequences, indirectly proving that S ₅ ⁿ is a non-functional gene.     

H-2-linked low-molecular weight polypeptide antigens assemble into an unusual macromolecular complex

The major histocompatibility complex (MHC) is a cluster of tightly linked genes whose products are of central importance in the functioning of the immune system. Class I and II MHC antigens are integral membrane proteins which regulate cell-surface interactions between T cells and their targets, while class III antigens are components of the complement system of serum proteins. All available evidence indicates that the structure and function of the MHC and its gene products are highly conserved among species (for review, see ref.5). We recently reported the existence in murine cells of a fourth class of MHC-linked polypeptides which are biochemically and genetically distinct from previously identified MHC gene products: BALB.B anti-BALB/c (anti-H-2d) antiserum immunoprecipitates a set of 16 cytoplasmic low-molecular weight polypeptides (LMP) from BALB/c spleen cells and from the WEHI-3 cell line. The production of these peptides is coordinately regulated (by immune interferon) with the production of the class I and II MHC antigens, suggesting that they too are functionally relevant to the immune system. We demonstrate here that these 16 polypeptides are associated with one another in vivo as a very large (580,000-molecular weight, Mr) noncovalent complex. The unusual nature of this complex has allowed the non-immunochemical identification of similar complexes from (serologically negative) H-2b murine cells and from a human cell line. Thus, LMP antigens display two properties in common with other MHC antigens: they are both polymorphic and genetically conserved across species.     

Correlation between a DNA restriction fragment length polymorphism and C4A6 protein

The fourth component of complement (C4) in man, is coded for by two separate but closely linked loci (C4A and C4B) within the major histocompatibility region (MHC), on the short arm of chromosome 6. Like class I and II loci of this region, the C4 genes are highly polymorphic with more than 30 alleles, including null alleles, assigned to the two loci. This extensive polymorphism, based mainly on electrophoretic mobility, provides a useful marker for studies of disease susceptibility. Several disorders, including systemic lupus erythematosus and type I diabetes, show associations with C4 phenotypes. We have used the technique of Southern with a C4 specific probe to examine the genomic DNA of individuals typed for C4 by protein electrophoresis. We have identified 10.7 and 3.8 kilobase (kb) BglII restriction fragments in each of 9 unrelated individuals with a C4A6 allele, and in none of 22 unrelated individuals in whom this allele was not expressed. This clear correlation of restriction fragment length polymorphism with C4 phenotype provides a precise basis for analysis of C4 polymorphism. It is likely to be of value in clinical investigations of autoimmune disease.