Structural relationship of the SB beta-chain gene to HLA-D-region genes and murine I-region genes

The major histocompatibility complex (MHC) regulates several aspects of the immune response. Class II antigens of the MHC control cellular interactions between lymphocytes. In man, at least three class II antigens (DR, DC and SB), consisting of distinct alpha- and beta-chains, are encoded in the HLA complex. Sequence analysis has established that the DR and DC antigens are the respective structural counterparts of the murine I-E and I-A antigens. Molecular cloning of the SB beta-chain gene has now enabled us to define its relationship to other class II genes. The DR, DC and SB beta genes have diverged from each other to the same extent. In murine DNA and in cloned genes from the I region, the best hybridization of SB beta DNA is with the E beta 2 sequence. E beta 2 may belong to a complete gene (E' beta) because first domain sequences were found adjacent to it.     

Isolation of a cDNA clone coding for an SB beta-chain

Class II antigens of the major histocompatibility complex (MHC) consist of a family of closely related cell surface-expressed glycoproteins. These antigens, which are genetically polymorphic, control important aspects of the immune response. At least three types of human class II antigens, namely, DR, DC and SB (refs 2-4), have been identified. All class II antigens are heterodimers composed of one alpha- and one beta-chain. The genes for both types of subunits are encompassed within the MHC. The general features of the DC and DR antigens have recently been elucidated. Much less is known, however, about the SB molecules. Here we describe the isolation of a cDNA clone as well as a genomic clone encoding a beta-chain whose amino acid sequence is compatible with the partial amino-terminal sequence of SB beta-chains.     

Structural and evolutionary analysis of HLA-D-region products

The major histocompatibility complex (MHC)--HLA in man and H-2 in mouse--encodes two classes of cell-surface antigens involved in the immune response. The amino acid sequences have been determined for a number of these molecules. Class I antigens, typified by the HLA-ABC antigens, are composed of a 43,000-molecular weight (MW) glycosylated transmembrane polypeptide with three external domains (alpha 1, alpha 2 and alpha 3), of which the one nearest the membrane (alpha 3) is associated with a 12,000-MW nonglycosylated polypeptide, beta 2-microglobulin. The HLA-D-region or class II antigens, DR, DC and SB, are composed of two glycosylated transmembrane polypeptides, of MWs 34,000 (alpha-chain) and 28,000 (beta-chain). Both chains have two external domains which presumably associate with each other, alpha 2, beta 2 being membrane proximal and alpha 1, beta 1 N-terminal and membrane distal. All four membrane-proximal domains (class I alpha 3, beta 2-microglobulin, class II alpha 2 and beta 2) have amino acid sequences that show significant similarities with immunoglobulin constant-region domains. This, together with the similarly placed internal disulphide bonds, suggests they might have an immunoglobulin-like structure (Fig. 1). We have now used computer graphics techniques to predict a detailed three-dimensional structure for the membrane-proximal domains of the class II antigens (alpha 2 and beta 2) based on the known coordinates of immunoglobulin constant domains (Fig. 2). The transmembrane regions of class II antigens have been modelled as two alpha-helices packed together. The proposed structure accounts for conservation of amino acids and leads to evolutionary predictions.     

A minimum of four human class II alpha-chain genes are encoded in the HLA region of chromosome 6

The major histocompatibility complex (MHC) in man, also called the HLA region, is located on the short arm of chromosome 6 and encodes antigens involved in immunological processes. The class II HLA antigens consist of two noncovalently associated polypeptide chains, one of molecular weight 34,000 (alpha) and the other of molecular weight 29,000 (beta). The extensive polymorphism of the beta chain(s) has allowed the genetic mapping of the corresponding beta gene(s) to the HLA-DR region. cDNA clones for the HLA-DR alpha chain have been used to map the non-polymorphic DR alpha-chain gene to chromosome 6 using mouse-human somatic cell hybrids. Similarly, the DR alpha-chain gene has been mapped to the short arm of chromosome 6 centromeric to the HLA-A, -B and -C loci by in situ hybridization experiments. We isolated a cDNA clone that is related to the DR alpha chain and encodes the class II antigen DC alpha chain. We describe here how this DC alpha clone was used to find two or three additional alpha-chain genes by cross-hybridization and how HLA-antigen loss mutants of a human lymphoblastoid cell line (LCL) were used to ascertain that these additional class II antigen alpha-chain genes are also located in the HLA region.     

The heavy chain of human B-cell alloantigen HLA-DS has a variable N-terminal region and a constant immunoglobulin-like region

The HLA-D region of the major histocompatibility complex (MHC) of man encodes polymorphic glycoproteins found predominantly on the cell surfaces of B cells and macrophages. These proteins mediate interactions, required for the induction of immune responses, among cells of the immune system and consequently are referred to as Ia (immune-response associated). Two families of Ia molecules, DR and DS (also known as DC), have been defined, the former analogous to the I-E (ref. 1) and the latter to the I-A molecules of the murine MHC. Both DR and DS molecules consist of two noncovalently associated polypeptide chains with molecular weights of 33,000 and 28,000, designated alpha and beta, respectively. The polymorphism of DR molecules is due to structural variation in the small subunit, DR beta, with the large subunit, DR alpha, being constant in structure. In contrast, both subunits DS alpha and DS beta are structurally variable when DS allotypes are compared. We have now isolated a cDNA clone from a DR7 cell line that contains the entire coding sequence for the DS alpha subunit and have compared its predicted amino acid sequence with that previously deduced from a DS alpha cDNA clone isolated from a DR4,w6 cell line. This comparison reveals that 10 of 11 amino acid differences are located within the alpha 1 (N-terminal) domain and that the alpha 2 or immunoglobulin-like domains are identical.     

A hybrid recognition sequence in a recombinant restriction enzyme and the evolution of DNA sequence specificity

Early attempts to generate new restriction specificities by recombination between allelic restriction-modification systems have been unsuccessful. Bullas et al. succeeded in isolating a new specificity, SQ, in Salmonella that they interpreted as being the result of a recombination event between the parental strains, Salmonella typhimurium and S. postdam, which encode the SB and SP restriction systems, respectively. This interpretation has recently been confirmed by DNA heteroduplex studies with the SB, SP and SQ structural genes. We have determined the DNA sequences recognized by the SB and SP enzymes and found that, like all type I restriction sequences, they are split into two specific domains by a spacer of nonspecific sequence that, for both SB and SP, is 6 base pairs (bp) long. We have now determined the sequence recognized by the recombinant SQ enzyme and find that it is a hybrid between the SB and SP sequences, containing one specific domain from each parental strain. This result implies that each of the two specific domains is recognized by a physically distinct part of the enzyme.     

The alpha 1 domain of the HLA-DR molecule is essential for high-affinity binding of the toxic shock syndrome toxin-1

Several exoproteins from the bacterium Staphylococcus aureus are highly potent polyclonal activators of T cells in the presence of cells bearing class II antigens of the major histocompatibility complex (MHC). These toxins, including the toxic shock syndrome toxin (TSST-1), act at nanomolar concentrations, bind directly to class II molecules, and do not require the processing typical of nominal antigen. Each toxin is capable of stimulating a subpopulation of peripheral T lymphocytes bearing particular V beta sequences as part of their alpha beta T-cell receptors. It is not known how these so-called 'superantigens' bind to class II and how this binding stimulates T cells. In this study, the different affinities of TSST-1 for human class II molecules DR and DP were exploited to define the region of a class II molecule necessary for high-affinity binding. Using chimaeric alpha- and beta-chains of DR and DP expressed at the surface of transfected murine fibroblasts and a binding assay with TSST-1, it was shown that the alpha 1 domain of DR is essential for high-affinity binding, and further that TSST-1 binding did not prevent subsequent binding of a DR-restricted antigenic peptide. This is compatible with a model of superantigen making external contacts with both class II and T cell receptor, and suggests that the V beta portion of the T-cell receptor interacts with the nonpolymorphic alpha-chain of DR.     

A combination of a particular HLA-DP beta allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease

Coeliac disease is an autoimmune disease of the intestinal mucosa, elicited by ingestion of wheat gluten in genetically susceptible individuals. Susceptibility to coeliac disease has been associated with the serologically defined variants DR3 and DR7 of the class II antigens encoded by the HLA-D region. Three related class II antigens, each consisting of an alpha and a beta glycoprotein chain, have been identified and are designated HLA-DR, HLA-DQ, and HLA-DP. These highly polymorphic transmembrane proteins bind peptides derived from the processing of foreign antigens and present them to T lymphocytes; they also influence the specificity of the mature T-cell repertoire. The role of HLA-DP polymorphism in susceptibility has not been as fully explored as that of the other class II antigens because of the complexity of the primed lymphocyte typing (PLT) method for determining DPw specificities. Here we use a new DNA-based method of HLA-DP typing to analyse the distribution of DP beta alleles in a group of coeliac disease patients and healthy controls. Two specific DP beta alleles (DPB4.2 and DPB3) are increased in the patient population. Comparison of the DP beta sequences suggests that the polymorphic residues at position 69 and at 56 and 57 may be critical in conferring susceptibility. Further, the contribution of the susceptible DP beta alleles appears to be independent of linkage to the previously reported DR3 and DR7 markers for coeliac disease. The distribution of DQ alpha and beta alleles in patients suggests that a specific DQ heterodimer may be responsible for the observed DR associations. Individuals with both this DQ antigen and a specific DP beta allele are at increased risk for coeliac disease.     

Isolation of an excision product of T-cell receptor alpha-chain gene rearrangements

The genes for the T-cell receptor, like the immunoglobulin genes, are rearranged as DNA. The mechanism of this rearrangement is not clear; unequal crossover between chromosomes and the looping-out and excision of the excess DNA have both been suggested. We isolated small polydisperse circular (spc) DNAs from mouse thymocytes and cloned them into a phage vector. Of the 56 clones we analysed, nine contained sequences homologous to T-cell receptor alpha-chain joining (J alpha) segments. We have characterized one of these clones; it contains one J alpha segment, and the product out of the recombination of a variable region of the alpha-chain gene (V alpha) with a J alpha gene segment. This is the first demonstration of the presence in extrachromosomal DNA of a reciprocal recombination product of any rearranging immunoglobulin or T-cell receptor gene. The finding verifies that V alpha-J alpha joining can occur by the looping-out and excision of chromosomal DNA.     

SB-restricted presentation of influenza and herpes simplex virus antigens to human T-lymphocyte clones

The HLA-D region of the human major histocompatibility complex (MHC) has been shown to be homologous to the murine I region in terms of both structure and function. Both regions encode class II MHC molecules which restrict T-lymphocyte interactions with antigen-presenting cells. We have recently described the MHC restriction and antigen specificities of human T-lymphocyte clones directed at strain A influenza virus. The majority of T-lymphocyte clones recognized antigen in the context of cell surface interaction products encoded by HLA-D/DR genes. However, a few clones recognized antigen presented by cells histoincompatible for D/DR antigens. We report here that some of these clones recognized viral antigens in association with antigens encoded by genes identical with or closely linked to the recently described secondary B-cell (SB) locus of the MHC. This is the first report that SB-restricted antigen recognition may form an integral part of normal, human immune responses.     

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.     

The changes in telomerase activity and telomere length in HeLa cells undergoing apoptosis induced by sodium butyrate

The changes in telomerase activity and telomere length during apoptosis in HeLa cells as induced by sodium butyrate (SB) have been studied. After a 48 h SB treatment, HeLa cells demonstrated characteristic apoptotic hallmarks including chromatin condensation, formation of apoptotic bodies and DNA Laddering which were caused by the cleavage and degradation of DNA between nucleosomes. There were no significant changes in telomerase activity of apoptotic cells, while the telomere length shortened markedly. In the meanwhile, cells became more susceptible to apoptotic stimuli and telomere became more vulnerable to degradation after telomerase activity was inhibited. All the results suggest that the apoptosis induced by SB is closely related to telomere shortening, while telomerase enhances resistance of HeLa cells to apoptotic stimuli by protecting telomere.     

Structure of the pro alpha 2 (I) collagen gene

Fifty-four kilobase pairs (kbp) of cloned chicken DNA containing the entire 38-kbp pro alpha 2 (I) collagen gene have been isolated and characterized. DNA sequence analysis of a select 4 kbp of the gene has precisely described 14 exons which comprise one-third of the sequences encoding the triple-helical domain of the collagen protein. These exons range in size from 45 to 108 base pairs (bp), are all multiples of the 9 bp that code for the repeating triplet, Gly-X-Y, and have an average size of 70 bp. About 50 introns interrupt this gene. Nevertheless, introns do not separate the coding sequences for the ends of the central triple-helical structural domain and the ends of the propeptide domains.     

Total volatile organic compound concentration and its influencing factors in urban indoor air after decoration

The indoor total volatile organic compound (TVOC) concentrations and their relationship to potential influencing factors were evaluated. Samples (n = 2302) were collected from 2007 to 2009 both in old structures and new construction in the city of Hangzhou from bedrooms, sitting rooms and studies that had been decorated within the previous year. The average TVOC concentration in all the newly decorated rooms was 0.65±0.69 mg/m 3 . 35.8% of samples exceeded the China standard. Over the past 3 years, the TVOC...     

Sequence polymorphism of HLA DR beta 1 alleles relating to T-cell-recognized determinants

HLA class II molecules are a highly polymorphic family of dimeric cell-surface proteins primarily involved in regulating T-cell responses to extrinsic antigens. To define regions of class II molecules involved in T-cell recognition, we have now compared sequences of three HLA DR beta cDNA clones obtained from cells that all express the same serologically defined determinants but differ in terms of T-cell-recognized specificities. The comparisons indicate that very few (one to four) nucleotides differ between what are almost certainly alleles of the DR beta 1 locus. All differences were in the first domain of the molecule and all localized to a region from amino acids 71-86. Because all differences were found only in this region of the molecule, and because DR alpha-chains seem to be relatively non-polymorphic, these positions in the DR beta-chain must have a major role in influencing T-cell recognition of the DR molecule.     

Polymorphism of human Ia antigens generated by reciprocal intergenic exchange between two DR beta loci

Class II molecules encoded by the human major histocompatibility complex (MHC) are involved in regulating T-cell response to antigens. The mechanisms for generating polymorphism in products of the MHC have been studied extensively for both the murine H-2 and the human HLA complex. Such studies indicate that point mutations plus selection have a major role in the generation of polymorphisms of class I and class II MHC genes. However, a non-reciprocal gene conversion mechanism has been proposed to explain several examples of clustered sequence variation in MHC genes. In all these examples, the proposed gene conversion event is unidirectional; that is, one of the two interacting genes acts as sequence donor and the other as sequence recipient. No examples of potential reciprocal genetic exchange (as occurs in the fungal system), in which the two interacting genes act as both donor and recipient of gene fragments, have been found in the MHC system or in other multigene families of higher organisms. We sequenced two different HLA-DR beta complementary DNAs from each of two different cells all expressing the same serologically defined determinant (DR2) but different T-cell-recognized (Dw) specificities (Dw12 and MN2). Sequence comparisons of these four cDNA clones (and two DR beta amino-acid sequences from the DR2-Dw2 subtype) suggest that new coding sequences for DR beta molecules in the DR2 haplotypes are potentially generated by reciprocal intergenic exchange.