Showing papers in "Immunogenetics in 1988"

A rat antibody against a structure functionally related to the mouse T-cell receptor/T3 complex.

Abstract: A monoclonal antibody KT3 (IgG2a, X) has been established from a fusion between NSO myeloma cells and spleen cells of a Sprague-Dawley rat hyper immunized with CBAT6 thymocytes by a method described previously (Tomonari 1985). KT3 was coupled to fluorescein isothiocyanate (FITC), and the tissue distribution in C57BL/10 mice was examined with a fluorescein-activated cell sorter (FACS). KT3 + cells were found as follows (mean percentage of three mice -+ SD): 37.7 + 0.8% of thymocytes, 40.3 -+ 1.5% of spleen cells, 63.2 +_ 1.8% of lymph node cells, and 4.8 -+ 1.1% of bone marrow cells. Al l strains of mice so far examined had similar percentages of KT3 + cells (data not shown). Nude mice (CBA) were the exception: KT3 + cells in spleen were only 0.6 -+ 0 .6%. KT3 activated spleen cells to proliferate in the absence and thymocytes to proliferate in the presence of phorbol myristate acetate (PMA) (Fig. 1). Induction of cytotoxicity by KT3 was then examined. T-cell receptor/T3 (Tcr/T3)-specific antibodies on target cells can tr igger cytotoxic activity by T cells (Lancki et al. 1984). Hybridoma cells secreting KT3 were specifically killed by T-cell clones C6 and D13 specific for K k + H-Y and K k + nonH-2, respectively, but a hybridoma (KT1) from the same fusion secreting an antibody irrelevant to the Tcr/T3 complex was not killed (Fig. 2A). Nonspecific cytotoxicity

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes.

Abstract: The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO βand DR αgenes, at least three DR βgenes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ α 1 β 1 to DQ α 2 β 4, possessed a single DQ α and a single DQ βgene whereas both these genes were duplicated in eight other haplotypes, DQ α 3 β 5 to DQ α 9 β 12. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQα DQ β , DRα DR β , and DO β restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions.

Abstract: Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO β , DQ α , DQ β , DR α and DR β genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ α , DY α , and DY β , are reported. DZ α was assumed to correspond to the human DZ α gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ α , DQ β , DR α and DR β loci and the other one is composed of the DO β DY α , DY β , and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational “hot spot” in the bovine class II region.

T-cell receptor-specific monoclonal antibodies against a V beta 11-positive mouse T-cell clone.

Abstract: Two monoclonal antibodies specific for the mouse T-cell receptor (Tcr) have been established by immunization with a Vβ11+ T-cell clone, clone C6. One is a rat antibody, KT11 (IgG2b, k), specific for the Vβ chain of C6, Vβ11. This was demonstrated by the fact that the strain distribution pattern of KT11+ cells was similar to that of Vβ5, 8, 9, 11, 12, and 13 and that the gene that encodes the molecule detected by KT11 was closely linked to Vβ8 in (B10 × SJL)F1 × SJL backcross mice. Furthermore, Vβ of C6 has been cloned from a λgt10 cDNA library and was demonstrated to be identical to the Vβ11 published sequences. All strains of mice that do not express major histocompatibility complex class II E molecules had higher numbers of KT11− cells than E+ strains. The KT11+ population in A strain mice and its H-2 congenic strains, however, was not affected by the presence or absence of E molecules. The other is a mouse antibody, KTL2 (IgM), specific for the idiotope of the Tcr expressed on the clone C6. Both antibodies were mitogenic and induced cytotoxicity. Expression of epitopes detected by KT11 or KTL2 was down-modulated by a T3e-specific antibody 145-2C11.

Biochemical complexity of serum HLA class I molecules

Abstract: Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around Mr 44,000, 40,000, and 35,000-37,000. HLA-A24-positive individuals are distinguished by higher serum levels of Mr 44,000 and 40,000 class I-like molecules than those found in HLA-A24-negative individuals. The Mr 44,000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The Mr 35,000 and 37,000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum Mr 44,000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The Mr 40,000 molecules do not contain a TM region. Mr 40,000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.

Genetic basis of ultraviolet-B effects on contact hypersensitivity.

Abstract: The genetic basis of the effects of ultraviolet B(UVB) radiation on the induction of contact hypersensitivity (CH) to dinitrofluorobenzene (DNFB) has been explored in genetically defined mice. It was found that acute, low-dose UVB radiation produced profound depletion of epidermal Langerhans cells (LC) at UVB-treated sites in all strains of mice tested. However, when DNFB was applied to UVB radiation sites, unresponsiveness developed in some strains of mice, but vigorous contact hypersensitivity was induced in others. The UVB-susceptible phenotype proved dominant or codominant in F1 hybrids derived from parental strains of the susceptible and UVB-resistant phenotypes. Experiments conducted in one set of F1 hybrids derived from two UVB-susceptible parental strains displayed UVB resistance, suggesting gene complementation, and showed that more than one genetic locus was involved. Segregant backcross populations, analyzed for the capacity to develop CH after UVB treatment and skin painting with DNFB, revealed that at least two, and probably three, independent genetic loci participate in determining UVB resistance. Results of experiments with H-2 congenic and recombinant mice derived from the B10 background implicated class I genes of the major histocompatibility complex as relevant genetic factors. These results indicate that there is a dissociation between the effects of UVB radiation on epidermal Langerhans cells and the capacity of a cutaneous surface to support the induction of contact hypersensitivity. The data indicate that the induction of CH to haptens is dependent on normal numbers of functional LC at the skin painting site only in some strains of mice. The data imply that in the so-called UVB-resistant strains of mice, alternative (non-Langerhans cell-dependent) mechanisms allow for the induction of CH. Several independent genetic loci, one of which appears to be H-2, govern this UVB-related effect.

Structural heterogeneity of human Pgp-1 and its relationship with p85.

Abstract: Pgp-1 is a major cell-surface phosphoglycoprotein (relative mass approximately 95 000) that has been identified in mouse and human cell lines and tissues (Isacke et al. 1986, Trowbridge et al. 1982, Hughes et al. 1983). Although monoclonal antibodies (mAb) that recognize this glycoprotein were initially raised against plasma membranes from NIH/3T3 fibroblast cells (Hughes and August 1981), its designation of Pgp-1 (phagocyte glycoprotein-1) was based on its marked abundance on macrophages, monocytes, and polymorphonuclear cells (Colombatti et al. 1982). Despite that the function of Pgp-1 is unknown, the mouse homolog is a useful differentiation antigen in that it is expressed on prothymocytes in the bone marrow (Trowbridge et al. 1982) and on a proportion of mouse thymocytes, including cells thought to be the immediate descendants of bone marrow prothymocytes (Lesley et al. 1985a, Hyman et al. 1986). Many cells found in an adult mouse thymocyte subpopulation enriched in Pgp-1 ÷ cells contain an unrearranged Tcell antigen receptor B-chain gene (Trowbridge et al. 1985). Further, Pgp-1 is expressed on more than 90% of fetal thymocytes on days 13 and 14 of gestation before the fraction of Pgp1 ÷ cells declines to adult levels shortly before birth (Lesley et al. 1985b). In contrast to the mouse, 50-60 % of human thymocytes express Pgp-1. The Pgp-1 ÷ subpopulation in the human comprises primarily medullary thymocytes, although some Pgp-1 ÷ cells scattered throughout the cortex can be detected by immunofluorescence staining (Isacke et al. 1986). p85 is a human glycoprotein with an approximate relative mass of 85 000 which was initially isolated from human chronic lymphocytic leukemia cells using mAb 50B4 and 50E6 (Letarte et al. 1985). More recently, the p85 glycoprotein was shown to carry all the antigenic deter-

HLA-DQ system and insulin-dependent diabetes mellitus in Japanese: does it contribute to the development of IDDM as it does in Caucasians?

Abstract: Fifty-six unrelated Japanese patients with insulin-dependent diabetes mellitus (IDDM) were HLA-typed, and restriction fragment length polymorphism (RFLP) analysis was performed after enzyme digestion with Bam HI and Taq I by using both DR and DQ probes. As previously reported, increased frequencies of Bw54, Cw1, DR4, and DRw53, which are in strong linkage disequilibrium in the Japanese population and make the characteristic Japanese haplotype, were confirmed. DQw4, a new allele of the DQ system recognized by the monoclonal antibody HU-46 and in linkage disequilibrium with this haplotype, presented the highest IDDM association. The RFLP analysis also showed the strongest correlation to IDDM when the DQ probe was applied. These results indicate that HLA-DQ might play the most important role in the development of IDDM in Japanese as well as in Caucasians. The correlation of DQβ amino acid sequences strongly associated with IDDM in Japanese are discussed in this study, and contrasting results were found when such sequences were compared with those of Caucasians.

Association between BoLA and subclinical bovine leukemia virus infection in a herd of Holstein-Friesian cows.

Abstract: The role of the bovine major histocompatibility system (BoLA) in subclinical bovine leukemia virus (BLV) infection was investigated in a herd of Holstein-Friesian cows (n = 240). The BoLA W8.1 allele was negatively associated with the presence of antibodies to the major BLV envelope glycoprotein, BLV-gp51 (corrected P less than 0.001, relative risk = 0.31). These results suggest that a BoLA-linked gene(s) may influence the early spread of BLV infection. Since B cells are the primary target of BLV infection, we then determined the relationship between BoLA-A locus phenotypes and B-cell numbers in peripheral blood of seropositive and seronegative cows. There were no significant differences between BoLA-A alleles for any hematological parameter in seronegative cows. Seropositive cows with the W12.1 allele had significantly greater absolute numbers of lymphocytes per microliter and B cells per microliter than did seropositive cows with other BoLA-A phenotypes (P less than 0.01, respectively). The average effect associated with the W12.1 allele in BLV-infected cows was an increase of 2010 B cells per microliter of whole blood relative to BLV-infected cows with other BoLA-A phenotypes. These results demonstrate that susceptibility to the polyclonal expansion of BLV-infected B lymphocytes is associated with the W12.1 allele in Holstein-Friesian cattle. Compared with results of a previous study in a herd of Shorthorn cattle, it appears that resistance and susceptibility to subclinical progression of BLV infection are associated with different BoLA-A locus alleles in different cattle breeds.

A structural model for the location of the Rodgers and the Chido antigenic determinants and their correlation with the human complement component C4A/C4B isotypes.

Abstract: Chido (Ch) and Rodgers (Rg) are human plasma and red blood cell antigens which were defined by the alloantibodies anti-Ch (Harris et al. 1967, Middleton and Crookston 1972) and anti-Rg (Longster and Giles 1976). AntiRg and anti-Ch are IgG antibodies produced in patients lacking either Rg or Ch antigen, respectively, who had undergone blood transfusion. The genes coding for the Rg and Ch antigens were both mapped to the human leukocyte antigen (HLA) region by serological studies (Middleton et al. 1974, Giles et al. 1976). The structural genes coding for the fourth component of complement (C4) (Rittner et al. 1975, Tiesberg et al. 1976), and the complement components C2 (Fu et al. 1974) and factor B (Allen 1974), were also mapped to the HLA region. Biochemical, genetic, and serological studies revealed that Rg and Ch are part of the antigenic components of the two isotypes of C4, C4A and C4B (previously called C4F and C4S), respectively (O'Neill et al. 1978a, b). Subsequently it was shown that the Rg and Ch antigens were both located in the C4d region of C4A or C4B (Tilley et al. 1978). C4d is the factor I-mediated proteolytic degradation fragment of C4 that may be covalently linked to various cell surfaces (see Reid and Porter 1981 for a review). C4A and C4B are highly homologous proteins (Belt et al. 1984, 1985, Yu et al. 1986) having markedly different electrophoretic mobilities, hemolytic activities (Mauff et al. 1983, Sire and Cross 1986), and chemical reactivities (Isenman and Young 1984, 1986, Law et al. 1984, Dodds et al. 1986, Shifferli et al. 1987) (Table 1). The latter is due to the differential reactivities of the thiolester carbonyl group in the two classes of C4: C4A exhibits relatively higher covalent binding affinity to amino groups or peptide antigens

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

Abstract: 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.

Squid glycoproteins with structural similarities to Thy-1 and Ly-6 antigens.

Abstract: In an attempt to identify invertebrate homologs of Thy-1 antigen, the optic and central nervous tissue of squid was solubilized in deoxycholate and fractionated by lentil lectin affinity chromatography and gel filtration to yield small abundant glycoproteins. Material with biochemical similarities to Thy-1 was found and shown to consist of two glycoproteins that were ultimately purified using monoclonal antibody affinity columns. Both glycoproteins were sequenced to yield sequences of 84 residues for Sgp-1 and 92 residues for Sgp-2. The sequences were analyzed for similarities to Thy-1 and other Ig-related sequences, and Sgp-1 showed some similarities that were > 3 standard deviation units away from mean random scores when tested with the ALIGN program. However, the sequence patterns were not typical of Ig-related domains and the relationship of Sgp-1 to the Ig superfamily remains problematical. Sgp-2 showed no relationship to the Ig superfamily, but similarities to Ly-6 antigen sequences were noted that are in accord with an evolutionary relationship. The similarities included ten Cys residues in each sequence of which eight were matched in the best alignment given by the ALIGN program. Chemical evidence was obtained for glycophospholipid tails at the COOH-termini of Sgp-1 and Sgp-2 as is the case for Thy-1 and Ly-6 antigens.

The susceptibility to insulin-dependent diabetes mellitus is associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3,4 heterozygotes

Abstract: In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P=6 x 10−6): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P=0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P=0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes. Since recent works have shown that the structural gene for the monokine tumor necrosis factor alpha (TNF-α) is located between the HLA-B and C4 loci and that TNF-α might be of importance in IDDM pathogenesis, the hypothesis is put forward that the C4-associated IDDM susceptibility reflects linkage dis-equilibrium between the C4 gene and a gene controlling TNF-α production. The high relative risk for IDDM in HLA-DR3,4 heterozygotes might be explained by the combined action of IDDM-specific susceptibility genes on DR4 haplotypes and DR3-linked susceptibility genes associated with predisposition to autoimmunity.

Attempt to detect recombination between B-F and B-L genes within the chicken B complex by serological typing, in vitro MLR, and RFLP analyses.

Abstract: In search for recombinants within the chicken major histocompatibility B complex, 1155 animals from crosses between the congenic lines CB (B12) and CC (B4) were tested with alloantibodies and monoclonal antibodies for the B-F (class I), B-L (class II), and B-G (class IV) antigens and by mixed lymphocyte reaction. The absence of detectable recombination was confirmed by restriction fragment length polymorphism analysis with B-L β and B-F probes. Together with previous reports, this indicates that the distance between the B-F and B-L loci is below 0.01 centimorgan.

MHC class II sequences of an HLA-DR2 narcoleptic.

Abstract: Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 β chains, along with DQw1 α and β chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3′ untranslated region of one of the DR2 β genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 α or β from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQα. Partial protein sequences of both DQ α and β from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR β or DQ α/β sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.

A homozygous point mutation results in a stop codon in the C1q B-chain of a C1q-deficient individual

Abstract: Southern blot analysis of the B-chain genes in one of eight C1q-deficient individuals revealed an abnormal banding pattern The defect, which was homozygous, could be localized by restriction mapping to a single Taq I site within residue 150 in the coding region of the B-chain gene DNA sequencing across the site revealed a stop codon that would cause premature termination of the protein product No material corresponding to the A or C chains, or a truncated B chain, could be identified by antigenic analysis of the patient's serum, indicating that a complete B chain is required for secretion of a C1q molecule

Biochemical and genetic analysis of the OKa blood group antigen.

Abstract: The monoclonal antibody TRA-1-85 recognizes a cell surface antigen which is expressed by all human cell types tested, including red blood cells (RBCs), but not by mouse cells. All the human RBCs tested were TRA-1-85 positive except those with the rare phenotype Ok(a−). Oka is a blood group antigen of very high frequency and only three unrelated Ok(a−) people are known. The red cells of all three propositi were negative with the TRA-1-85 antibody. To confirm the relationship between the TRA-1-85 antibody and anti-Oka, the immune antibody found in the serum of Ok(a−) individuals, Western blot analysis was used: the TRA-1-85 antibody and anti-Oka gave identical but complex patterns of re-activity in Western blot analysis of human cell lysates or membranes. This suggests that the anti-Oka and TRA-1-85 antibodies recognize the same cell-surface determinant and implies that Oka is not restricted in its expression to the surface of RBCs but is expressed on white blood cells (WBCs) of Ok(a+) individuals and all human cell lines tested to date. WBCs from one of the Ok(a−) propositi were tested and found to be negative with the TRA-1-85 antibody. Finally, the species specificity of the TRA-1-85 antibody has been exploited by the use of somatic cell hybrids and DNA transfection techniques to examine the genetic control of the Oka antigen defined by the TRA-1-85 antibody. We report that the determinant is controlled by a single gene OK present on human chromosome 19.

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

Abstract: 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 λgt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, λbg28, 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 λbg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the λbg28 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 λbg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.

Regulation of Qa-1 expression and determinant modification by an H-2D-linked gene, Qdm.

Abstract: We examined the regulation of cell surface expression of the Qa-1 alloantigens using a panel of monoclonal anti-Qa-1 cytotoxic T-lymphocyte (mCTL) lines In contrast to previous reports of tissue-specific expression, we found that Qa-1 was widely expressed, resembling the prototypical class I H-2K/D molecules We further found that an H-2D-linked gene, which we termed Qdm for Qa-1 determinant modifier, controlled expression of certain CTL-defined Qa-1 antigenic determinants H-2D khomozygous haplotypes expressed a recessive allele of the modifier, Qdm k, whereas all other H-2 haplotypes tested expressed a dominant allele, Qdm + The Qdm + allele regulated in trans Qa-1 epitope expression from a Qdm kchromosome, modifying expression of particular CTL-defined Qa-1 antigenic determinants rather than affecting levels of cell surface expression Mechanisms of Qdm function may include either a novel protein modification system or an unprecedented case of antigen recognition restricted by a nonclassical major histocompatibility complex molecule

Class II genes of miniature swine. I. Class II gene characterization by RFLP and by isolation from a genomic library.

Abstract: Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human α probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human β probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLAc haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique α genes were obtained which showed no evidence of cross-hybridization, while β genes showed extensive cross-hybridization and were frequently detected in the library by more than one human β gene probe. These data are consistent with early evolutionary divergence of a genes, prior to mammalian speciation, and with continuing evolution of β genes, with possible shared usage of these genes by different a loci. The data also imply that α genes can readily be assigned to loci homologous to their human counterparts, but that β genes will require further mapping and/or sequence analysis to confirm assignments.

Sequence polymorphism of human complement factor H.

Abstract: Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.

Evidence for linkage between the caprine leucocyte antigen (CLA) system and susceptibility to CAE virus-induced arthritis in goats

Abstract: The distribution of caprine leucocyte antigens (CLA) in goats from four different breeds (n=546) affected by caprine arthritis-encephalitis virus (CAEV)-induced arthritis were determined and compared breed for breed with those of infected but clinically healthy controls (n=402). Differences in frequencies of some of the CLA specificities between the affected and control groups were found, but after correction of the ordinary P values for number of observed alleles, only the CLA Be7 specificity in the Saanen breed showed a significant deviation at the 0.05 probability level. Animals of the Saanen breed carrying this specificity are less prone to develop arthritis after CAE virus infection than goats lacking this specificity. Eleven groups (multiple-case families or halfsibling groups with at least two informative diseased offspring/group) were analyzed for manifestation of the disease and segregation of the parental haplotypes. The results of the maximum likelihood test of association (P<0.005) and the calculated high lod score value of 5.70 give evidence for linkage between the locus encoding the determined class I CLA alleles and a hypothetical locus (i) coding for genes responsible for arthritis resistance/susceptibility. The particular class I CLA allele associated with the disease susceptibility varied from family to family, however. These data provide the first evidence that CAE virus-induced arthritis in the goat is genetically influenced by the MHC system; they also suggest that susceptibility/resistance genes are not directly associated with the determined class I gene products but rather are in close genetic linkage.

Chromosomal assignment of the gene encoding the mouse tumor rejection antigen gp96.

Abstract: Tumor rejection antigens (TRAs) of chemically induced tumors of inbred mice have long been regarded as the prototype of tumor-specific antigens. These antigens were initially detected in studies showing that prior tumor growth renders mice resistant to subsequent tumor challenge (Foley 1953). An intriguing feature of these antigens is their extensive polymorphism: mice immunized with a particular tumor are resistant to subsequent challenges with that tumor, but not other tumors induced with the same carcinogen (Prehn and Main 1957). Even two tumors induced in one animal with the same carcinogen have been shown to be antigenically distinct (Globerson and Feldman 1964). We have recently demonstrated that the TRA activities of two antigenically distinct, chemically induced tumors copurify with glycoproteins of 96 000 d (gp96) (Srivastava et al. 1986). Purified gp96 from these two tumors share an identical 14-amino-acid sequence at their amino termini. Antiserum raised in rabbits against purified gp96 detects 96 000 d molecules in a variety of normal and malignant cell types. These observations have led us to propose that the individually specific antigenicity of chemically induced tumors is mediated by a family of gp96 molecules, which are structurally related but distinct in individual tumors. To provide a structural basis for the extensive polymorphism displayed by TRAs, genes encoding the gp96 molecules are being isolated from tumors and normal cells (Srivastava et al. 1987, Srivastava and Old 1988). In this report, we describe the chromosomal localization of the gp96 encoding genes by analyzing a panel of mouse x chinese hamster hybrids. The hybridization probe used to analyze Southern blots represents a DNA fragment pMA2 isolated from a cDNA library of the BALB/c Meth A sarcoma (Srivastava et al. 1987). This 311 basepair probe spans 5' untranslated region and exons 1, 2, and 3 of the gp96 gene. pMA2 was hybridized to

Characterization of a new subfamily of class I genes in the H-2 complex of the mouse.

Abstract: A previously undescribed subfamily of mouse class I MHC genes, consisting of two to three members, has been identified. The structure and organization of one of these, Mb1, has been determined. Mb1, consists of five exons with open reading frames and potentially encodes a class I-like transmembrane protein. In the genome, Mb1 is linked to the H-2 complex, mapping telomeric to Qa. However, this gene has low (ca. 60%) nucleotide identity with other class I sequences and is no more related to mouse class I genes than to class I genes from other species. Mb1 transcripts have not been found in a variety of adult tissues or cell lines, suggesting that, if Mb1 is expressed, its expression is highly regulated. From DNA sequence identity and intron-exon organization, Mb1 appears to be a primordial gene which antedates mouse speciation and which has evolved independently of the rest of the class I gene family. Examination of various species of wild mice demonstrates the presence of a discrete Mb1 subfamily over long evolutionary periods of time.

Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA class I and III regions by field inversion gel electrophoresis.

Abstract: Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA class field inversion gel electrophoresis 11HlllUllO-genetics The human major histocompatibility (HLA) complex is located on the short arm of chromosome 6 in the 6p21.31-6p21.33 region (Spring et al. 1985, Ziegler et al. 1985a). The physical length of the entire HLA complex is unknown so far, but our estimate based on the separation of DNA fragments containing HLA genes by pulsed-field gel electrophoresis (Schwartz and Cantor 1984, Carle and Olson 1984) indicates that it encompasses at least 2500 kb pairs (Ragoussis et al. 1986). This estimate has recently been confirmed (Lawrance et al. 1987). Apart from the highly polymorphic class I and II loci, the HLA complex contains the genes for several complement components and 21-steroid hydroxylase (class III region) (Lamm and Olaisen 1985). In addition, the loci for tumor necrosis factor (TNFA) as well as lymphotoxin (TNFB) are also near or even within the HLA region (Nedwin et al. 1985). Spies and co-workers (1986) demonstrated that the TNF genes map either centromeric to HLA-DP or telomeric to the class I1 region, although apparently not in the vicinity of any known class I or III genes. The recent demonstration that the TNF loci are situated 70 kb upstream of the H-2D gene in the BALB/c mouse between the class III and class I regions (Mfiller et al. 1987a, b) suggested an analogous location in man, because the genetic organization of the major histocompatibility complexes (MHC) of both species is very similar. To clarify the position of the TNFA and TNFB genes on the HLA map, we have assigned TNFA to large DNA restriction fragments separated by field inversion gel electrophoresis (FIGE) (Carle et al. 1986), which hybridize with either class 1II-or class I-specific probes as well. These results prove that the TNFA locus is localized between the HLA class III region and the HLA-B locus. To avoid interpretative difficulties which might arise from haplotype-specific restriction fragment length poly-morphisms, mutant human cell lines with monosomy 6 or HLA hemizygosity were employed. All mutants were derived from BJAB-B95.8.6 lymphoma cells with the HLA haplotypes A1, Cw4,B35 and A2, C-,B13 (Spring et al. 1985). Mutant BM 19.7 is a monosomy 6 mutant cell line retaining the A2 haplotype (Ziegler et al. 1985b). BM 28.7 also exhibits monosomy 6, but with loss of the chromosome bearing the A2 haplotype (Ragoussis et al. …

A novel HLA-DR beta I sequence from the DRw11 haplotype.

Abstract: The most striking feature of major histocompatibility complex (MHC)-encoded molecules is their extensive variability (for a thorough review see Klein 1986). The interaction of MHC proteins with T cells during antigen presentation and recognition is crucial for the course of the immune response (Klein 1986). The heterodimeric HLA-DR molecules are the most abundantly expressed MHC class II molecules in man. Their high degree of polymorphism is entirely due to the variability of the DR~ chains, where the variable amino acid residues are concentrated in the aminoterminal domain (Kaufman et al. 1984, Rask et al. 1985, Rollini et al. 1985). The identification of sequence differences is a prerequisite for the future understanding in molecular terms of the immune response in health and disease. In order to characterize the restriction element of a strictly autoreactive T-cell clone (UA-S2; Schlesier et al. 1987), the DNA sequence of the DRwllf l gene was determined from genomic and cDNA libraries (A. Hinkkanen, V. Steimle, M. Schlesier, H. H. Peter, and J. T. Epplen, submitted for publication). UA-S2 has been derived from the arthritic joint of an individual who typed homozygous DRw11/w11 (Schlesier et al. 1987). The libraries were screened for phages containing DR~ sequences with a DR~ cDNA probe (Gustafsson et al. 1984) as well as with a synthetic oligonucleotide, which is complementary to a nonpolymorphic region in the transmembrane part of the DR¢ gene (amino acid positions 199-206). Hybridizing inserts of phage clones were either subcloned into the pUC19 plasmid vector for restriction mapping or were directly subcloned into M13mp18/19 phages for sequencing by the chain-termination method (Sanger et al. 1980). Restriction mapping of the DR~positive genomic clones with exon-specific oligonucleotide probes revealed that the maps of six clones are completely identical to that of the DRw6fl locus (Gorski et al. 1987). Both the DRwll and the DRw6 haplotypes be-

Antigenic determinants expressed by human C4 allotypes; a study of 325 families provides evidence for the structural antigenic model

Abstract: The antigenic determinants of human C4 have been defined by human IgG antisera, Rodgers (Rg) and Chido (Ch), in hemagglutination-inhibition assays (HAI). Eight (2 Rg and 6 Ch) are of high frequency, > 90% , and 1, WH, is of low frequency, 15 %. The phenotypic combinations are complex; generally, C4A expresses Rg, and C4B has Ch, but reverse antigenicities have been established both by HAI and by sequence data of selected C4 allotypes. A study of 325 families provides data on the antigenic expression of each C4 allotype and demonstrates strong associations. A structural model for the antigenic determinants of C4 proteins has been proposed and is completely supported by the family material. Of the 16 possible antigenic combinations for C4 proteins, only 3 are undetected. A new Ch combination has been recorded in two French families. The reported sequence variation within the C4d region can account for the antigenic determinants but leaves the location of electrophoretic variation in C4 still unclear.

Chromosomal localization of the major histocompatibility complex of the horse (ELA) by in situ hybridization.

Abstract: The first gene assignment to a horse chromosome is reported for equine leucocyte antigen (ELA), the major histocompatibility complex of the horse. A cloned DNA sequence derived from a class I gene of the porcine major histocompatibility complex was used as a probe for an in situ hybridization experiment. We present the regional localization of ELA, using this sequence, to equine chromosome 20g14-q22.

Association of membranous nephropathy with T-cell receptor constant β chain and immunoglobulin heavy chain switch region polymorphisms

Abstract: We have investigated T-cell antigen receptor constant β chain genes (Tcr Cβ) and immunoglobulin (Ig) heavy chain switch region genes of HLA-DR-typed patients with membranous nephropathy (MN) employing DNA restriction fragment length polymorphism (RFLP) analysis. When a Tcr Cβ probe in conjunction with the restriction endonuclease BgI II was used, a significant increase in the frequency of a 10.0;9.2 kb heterozygous RFLP phenotype was found in MN (75.0 % versus 42.1 in controls; P=0.002). When Sst I-restricted DNA from MN patients was hybridized with a DNA probe homologous to the switch region flanking the Ig Cµ heavy chain gene (Sµ), there was a significant decrease in the frequency of the 2.1; 2.6 kb heterozygous RFLP phenotype in MN (24.0% versus 54.6% in controls; P=0.004). These results suggest that Tcr beta and Ig heavy chain loci, as well as HLA antigens, may be important in the pathogenesis of MN.

The human Vpre B gene is located on chromosome 22 near a cluster of V lambda gene segments.

Abstract: The chromosomal location of the human VpreB gene was determined by Southern blotting analysis of restriction enzyme-digested DNAs from a panel of 17 mouse-human somatic cell hybrids. The pattern of hybridization of a Vpre B-specific probe in conjunction with earlier analysis of several marker genes allowed the following conclusions: 1) Vpre B is on human chromosome 22 within band 22q11.2 distal to the bcr-like gene, bcr-2 and proximal to the bcr-like gene, bcr-4. 2) Vpre B has been localized relative to several constitutional and tumor-specific breakpoints within 22q11.2, segregates in hybrids retaining 22q- chromosomes with some but not with all members of the V lambda 1 subgroup of the V lambda genes, and is amplified with these genes in K562 cells. 3) The order of the loci on chromosome 22 is centromere----bcr-2, Vpre B, V lambda 1----bcr-4----C lambda----bcr-1----bcr-3----sis.