Histocompatibility antigens controlled by theI region of the murineH-2 complex

Spleen cells from A.TH mice, presensitized in vivo by skin grafting, were restimulated in vitro by A.TL lymphocytes, and A.TH anti-A.TL effector cells were generated. The effector cells lysed, in the CML assay, A.TL blasts. This reaction, which was againstI-region antigens, could be inhibited by the addition to the reaction mixture of anti-La sera directed against A.TL antigens. The inhibition was specific, since normal mouse serum, reciprocal antiserum (A.TL anti-A.TH), and anti-H-2 sera did not have a significant effect on the reaction. The Ia antibodies also specifically inhibited the reaction of A.TH anti-A.TL effector cells against CBA targets. Con A blasts were significantly poorer targets inI-region CML than LPS blasts. As CML targets, macrophages and cells of a mammary adenocarcinoma were as good as, if not better than, the LPS blasts. The experiments support the notion that Ia antigens are the targets in theI-region CML.     

Biochemical confirmation of recombination within the B-G subregion of the chicken major histocompatibility complex

Analysis of the B-G antigens of eight chicken major histocompatibility complex (B) system recombinant haplotypes by high resolution two-dimensional gel electrophoresis has provided evidence for the transfer of the complete B-G subregion in seven cases. In the eighth, a partial duplication within the B-G subregion appears to have occurred. In this recombinant, the entire array of polypeptides associated with one parental allele, B-G ²³ is expressed together with nearly the entire array of B-G polypeptides of the other parental haplotype, B ². This compound polypeptide pattern corroborates the serological evidence for a partial duplication within the B-G subregion and provides indirect evidence for the existence of multiple loci within B-G and for a means by which polymorphism may be introduced into the chicken major histocompatibility complex.     

Genotyping chickens for the B-G subregion of the major histocompatibility complex using restriction fragment length polymorphisms

Chicken B-G-subregion cDNA probes were used to analyze restriction fragment length polymorphisms (RFLP) of the B-G subregion of the chicken major histocompatibility complex. Genomic DNA from chickens representing 17 of the 27 standard B haplotypes were digested with restriction endonucleases and analyzed in Southern hybridizations with two cDNA clones from the B-G subregion. Each B-G genotype was found to produce a unique pattern of restriction fragments in these Southern hybridizations. With 15 of the 17 genotypes examined, the different genotypes could be readily distinguished in hybridizations produced with DNA digested with a single restriction enzyme, PVU II. The two additional genotypes produced nearly identical patterns in PVU II preparations and with three additional enzymes as well, but were readily distinguishable in Eco RI digestions. For many of the haplotypes, samples from several individuals in different flocks were examined. In every instance, genotyping by RFLP pattern was found to confirm the B-G allele assigned serologically.     

Restricted recognition of H-2 subregion coded alloantigens in delayed-type hypersensitivity

Subcutaneous (sc) immunization of mice with H-2K, I, or D incompatible spleen cells induces a state of host-versus-graft (HvG) delayed-type hypersensitivity (DTH). The DTH reaction is elicited by challenging the immunized mice in a hind foot with similar allogeneic spleen cells and is measured as the subsequent foot swelling. DTH effector T cells specific for H-2I-coded alloantigens, but not for H-2K/D-coded alloantigens, can be induced in a graft-versus-host (GvH) model as well. In this paper we report that under HvG as well as under GvH conditions the recognition of class II antigens by DTH effector T cells is restricted by class I molecules. Furthermore, DTH effector T cells induced by sc immunization with class I antigens appear to be restricted by class II molecules.     

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.     

A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis

The plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, gamma-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong fluorescence. To confirm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1-3 microm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them 'bulbs'. Three-dimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The fluorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give fluorescent signals of bulbs in transgenic plants, but the existence of bulbs was still confirmed by electron microscopy. These results suggest that bulbs define a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.     

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.     

A variant alpha-chain in an Ia molecule from the I-A subregion of the mouse major histocompatibility complex: a possible intragenic recombination

The recombinant MHC haplotype t1, present in mouse strain A.TL and B10.TL, encodes an I-A molecule with a variant alpha-chain that was recognized when tryptic peptide digests from t1 and the nominal parental haplotype (a1 or k) were compared by double-label reverse phase HPLC. The t1 I-A alpha-chain possesses a tryptic peptide absent from a1 and k I-A alpha-chains, and lacks several peptides present in a1 and k. The peptide that differs in t1 is not a mannose-bearing peptide, and thus its altered mobility is probably not due to carbohydrate modification. The alteration in t1 could result from mutation or intragenic recombination, but the unique t1 peptide migrates in a position identical to a peptide found in haplotype s, a partner in the (s X a1) cross that generated the t1 recombinant. If this apparently shared peptide indicates an intragenic recombination, this places the I-A alpha-gene centromeric to the other I-A subregion gene, and together with the data of Jones, suggests a gene order of I-A alpha, I-A beta, I-E beta (A alpha, A beta, Ae).     

The chimpanzee major histocompatibility complex class II DR subregion contains an unexpectedly high number of beta-chain genes

The major histocompatibility complex (MHC) class II DR subregion of the chimpanzee was studied by restriction fragment length polymorphism (RFLP) analysis. Genomic DNA obtained from a panel of 94 chimpanzees was digested with the restriction enzyme Taq I and hybridized with an HLA-DR probe specific for the 3' untranslated (UT) region. Such a screening revealed the existence of 14 distinct DRB/Taq I gene-associated fragments allowing the definition of 11 haplotypes. Segregation studies proved that the number of chimpanzee class II DRB/Taq I fragments is not constant and varies from three to six depending on the haplotype. Comparison of these data with a human reference panel manifested that some MHC DRB/Taq I fragments are shared by man and chimpanzee. Moreover, the number of HLA-DRB/Taq I gene-associated fragments detected in a panel of homozygous typing cells varies from one to three and corresponds with the number of HLA-DRB genes present for most haplotypes. However, a discrepancy is observed for the HLA-DR4,-DR7, and -DR9 haplotypes since a fourth HLA-DRB pseudogene present within these haplotypes lacks its 3' UT region and thus is not detected with the probe used. These results suggest that chimpanzees have a higher maximum number of DRB genes per haplotype than man. As a consequence, some chimpanzee haplotypes must show a dissimilar organization of the MHC DR subregion compared to their human equivalents. The implications of these findings are discussed in the context of the trans-species theory of MHC polymorphism. Address correspondence and offprint requests to: R. E. Bontrop.     

Mutation in the NDUFS4 gene of complex I abolishes cAMP-dependent activation of the complex in a child with fatal neurological syndrome

Evidence is presented showing that in a patient with fatal neurological syndrome, the homozygous 5 bp duplication in the cDNA of the NDUFS4 18 kDa subunit of complex I abolishes cAMP-dependent phosphorylation of this protein and activation of the complex. These findings show for the first time that human complex I is regulated via phosphorylation of the subunit encoded by the NDUFS4 gene.     

Comparative anatomy of the primate major histocompatibility complex DR subregion: evidence for combinations of DRB genes conserved across species.

The class II region of the human major histocompatibility complex (HLA) is made up of three major subregions designated DR, DQ, and DP. With the aim of gaining an insight into the evolution and stability of DR haplotypes, a total of 63 cosmid clones were isolated from the DR subregion (Gogo-DR) of a western lowland gorilla. All but one of these cosmid clones were found to fall into two clusters. The larger cluster, A, was defined by 41 overlapping cosmid clones and contained a DRB gene segment made up of exons 4 through 6 and four DRB genes, designated Gogo-DRB6, Gogo-DRB5*01, Gogo-DRB8, and Gogo-DRB3*01. The total length of this cluster was approximately 180 kb. The second cluster, B, encompassed a contiguous DNA stretch of approximately 145 kb and was composed of 21 overlapping cosmid clones. Cluster B contained three DRB genes, designated Gogo-DRB1*08, Gogo-DRB2, and Gogo-DRB3*02. One cosmid clone (WP1-9) containing a DRB pseudogene could not be linked to either cluster A or B. Neither the organization of cluster A nor that of cluster B was identical to that of known HLA-DR haplotypes. However, two gorilla DRB genes, Gogo-DRB6 and Gogo-DRB5*01, the human counterparts of which are linked in the HLA-DR2 haplotype, were found to be located next to each other in cluster A. The arrangement of the Gogo-DRB genes in cluster B, which is presumed to be the gorilla DR8 haplotype, was similar to that of HLA-DR3/DR5/DR6 haplotypes and to that of the presumed ancestral HLA-DR8 haplotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for more than one Ia antigenic specificity on molecules determined by the I-A subregion of the mouse major histocompatibility complex.

Ia antigenic specificities determined by the I-A subregion of the mouse major histocompatibility complex have been examined in strain B10.D2 (H-2d), C57BL/10 (H-2b), and in a (C57BL/6xDBA/2) hybrid (BDF1; H-2b/d). Detergent solubilized, 3H-leucine-labeled antigen preparations were mixed with appropriate alloantisera and precipitation was induced either by addition of goat anti-mouse gamma-globulin or by addition of protein A-bearing Staphylococci. Sequential precipitation analysis showed that in strain B10.D2, Ia specificities 8 and 11 were co-precipitable, and that in strain C57BL/10, Ia specificities 8 and 9 were co-precipitable. In contrast, precipitation of specificities 9 and 11 from a BDF1 antigen preparation showed that these two Ia specificities were on separate molecules. The genetic implications of these data are discussed.