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H T Orr

Publications and source records attributed to H T Orr.

At least 127 records · Page 7Linked to original sources
PubMed1985

Cloning and complete sequence of an HLA-A2 gene: analysis of two HLA-A alleles at the nucleotide level.

The gene for the HLA-A2 antigen has been cloned from the human lymphoblastoid cell line 721. Comparison of this sequence with the published sequence for HLA-A3 permits the examination of two alleles at the extremely polymorphic HLA-A locus. A high degree of sequence conservation was seen in both coding and noncoding DNA, 97.2% and 94.5%, respectively. Interestingly, the 3' untranslated region was the most conserved, with 99.5% homology. The polymorphism of the HLA-A antigens results from a high proportion of amino acid substitutions relative to the total nucleotide changes in exons 2 and 3. Unlike the clustered differences seen in this region on comparison of two H-2K alleles of mouse, nucleotide substitutions between the HLA-A2 and A3 alleles are evenly distributed. Substitutions at silent sites and within introns were used to calculate an intra-allelic divergence time of at least 10 to 15 million years for these two HLA-A alleles.

Alleles↗
PubMed1985

Restriction fragment length polymorphisms as markers of engraftment in allogeneic marrow transplantation.

We have used DNA hybridization techniques employing restriction fragment length polymorphisms (RFLPs) to quantitate the level of donor cell engraftment in bone marrow transplantation recipients. The genetic origin of the bone marrow cells and various peripheral blood populations was analyzed in 14 patients. We found at least one informative polymorphism for each donor-recipient pair. Additional markers of engraftment included cytogenetic analysis, HLA typing, and red cell typing. By DNA analysis, four patients had complete engraftment, five had partial engraftment, and five had no evidence of donor cell engraftment. In three cases, DNA analysis permitted detection of minor populations (5% to 10%) of donor or host cells. Eight of fourteen patients were evaluable for chimerism posttransplant by cytogenetic analysis. In five cases, cytogenetic results were completely concordant with DNA analyses. In two cases of apparent autologous recovery, as assessed using RFLPs, a small number of cells of donor karyotype was seen. In one other case, a small number of cells of host karyotype was not detected by RFLP studies. HLA typing in three partially engrafted patients was purely either of donor or host type. Red cell typing was discordant with DNA and/or cytogenetic results in four of eight cases. We conclude that DNA analysis at a limited number of informative genetic loci is useful for quantitating the degree of engraftment in multiple populations of nondividing cells following allogeneic bone marrow transplantation.

Adolescent↗
PubMed1984

Isolation of HLA locus-specific DNA probes from the 3'-untranslated region.

When human class I cDNA clones containing coding sequences are used to probe genomic DNA, 15-20 fragments, each containing a complete class I gene or pseudogene, are seen. Identification of which genomic DNA segments encode the HLA-A and -B antigens has to date required transfection of mouse L cells with cloned class I genes or analysis of HLA loss mutants. In this report we show that under high-stringency conditions, probes constructed from the 3'-untranslated region can be used to specifically identify the segments of DNA that encode the HLA-A and -B antigens in the human lymphoblastoid cell line 721. Examination of DNA from unrelated individuals indicates that these probes are locus specific and will permit identification of HLA-A and -B genes in the population.

Cell Line↗
PubMed1983

Class I-like HLA genes map telomeric to the HLA-A2 locus in human cells.

Among the proteins encoded by the major histocompatibility complex (MHC) are the highly polymorphic class I major transplantation antigens, HLA-A and HLA-B in man and H-2K and H-2D in mouse. Class I loci also include the less polymorphic HLA-C in man and H-2L in mouse. Class I antigens are 45,000-molecular weight (Mr) glycosylated membrane proteins associated on the cell surface with apparently nonpolymorphic beta 2-microglobulin. Linked to the murine MHC are genes encoding the class I-like Qa and Tla antigens, which are closely related structurally to H-2K, D and L, as they are also 45,000-Mr cell surface glycoproteins associated with beta 2-microglobulin. Such class I molecules have also been found on the surface of human T lymphocytes and, in one case, shown to be linked to HLA-A. Recombinant DNA techniques have been used to map DNA fragments to the Tla region in mouse. A similar genetic analysis of the HLA complex is hampered by the lack of congeneic strains and by the small number of intra-MHC recombinants in well studied families. One means of overcoming these problems involves a molecular genetic analysis of human lymphoblastoid cell line (LCL) mutants having gamma-ray-induced physical deletions of HLA DNA to map DNA fragments in the MHC. Here we have applied this method to provide evidence that some human class I-like DNA sequences are telomeric to the A2 locus in LCL 721.

Cell Line↗
PubMed1983

Mapping of class I DNA sequences within the human major histocompatibility complex.

Mutants that had lost expression of alleles of one or more HLA loci were isolated with immunoselection after gamma-irradiation of a human lymphoblastoid cell line LCL 721. DNAs from the mutants were digested with restriction endonucleases and analyzed by Southern blotting using probes for class I HLA genes. Eight polymorphic cut sites for HindIII and PvuII were discovered in class I-associated sequences of LCL 721. Losses of specific fragments generated by restriction enzymes could be associated with losses of specific antigenic expressions and it was possible in this way to assign HLA-A1, HLA-A2, and HLA-B8 to specific DNA fragments. Patterns of gamma-ray-induced segregations of DNA fragments permitted rough linkage alignment of about 30% of the fragments generated by PvuII. The resultant map showed that there are class I HLA genes on the telomeric side of the HLA-A locus. Restriction enzyme site polymorphisms were also examined in a panel of DNAs isolated from peripheral blood lymphocytes (PBLs) of HLA-typed individuals. This panel of PBL DNA complemented the analysis using the HLA deletion mutants.

Alleles↗
PubMed1982

Structure of crossreactive human histocompatibility antigens HLA-A28 and HLA-A2: possible implications for the generation of HLA polymorphism.

The primary structure of two highly crossreactive human histocompatibility antigens, HLA-A28 and HLA-A2, has been determined to 96% and 90%, respectively, of the papain-solubilized molecules. Their sequences have been compared with the sequence of HLA-B7 and with each other in order to outline the sites of diversity. The overall homology between HLA-B7 and these HLA-A antigens is 86%. A large majority of the differences are located between residues 43 and 195. Within this area, substitutions cluster in at least three segments--residues 65-80, 105-116, and 177-194. HLA-A28 and HLA-A2 show 96% homology. Most of the differences fall within segments 65-74 and 107-116. These results strongly support the suggestion that residues in these segments are integral parts of the alloantigenic determinants of HLA-A28 and HLA-A2. It is further proposed that these three clusters may constitute major, albeit not exclusive, sites of antigenic diversity in human histocompatibility antigens. The nature of the differences among HLA-B7, HLA-A28, and HLA-A2 in the first variable segment suggests that gene conversion might play some role in the generation of HLA polymorphism.

Amino Acid Sequence↗
PubMed1981

Biosynthesis and cell surface localization of nonglycosylated human histocompatibility antigens.

The effects of the inhibitor of N-linked glycosylation, tunicamycin, on the synthesis of HLA-A and -B antigens in the human lymphoblastoid cell line JY are described. HLA-A and -B antigens are membrane glycoproteins that consist of a two chain complex, the heavy chain being glycosylated at Asn 86, whereas the light chain, identical to beta 2-microglobulin, is not glycosylated. HLA-A and -B antigens synthesized in the presence of the antibiotic are devoid of carbohydrate. This lack of carbohydrate does not affect the association of the heavy and light chains, nor does it affect the reactivity with human alloantisera, or a mouse monoclonal antibody W6/32, that reacts with all HLA-A and -B specificities examined so far. Nonglycosylated HLA-A and -B antigens are no more susceptible to proteolysis with trypsin, chymotrypsin, or papain than their fully glycosylated counterparts. Thus it may be concluded that the carbohydrate side chains of HLA-A and -B antigens do not contribute significantly to the conformation of HLA-A and -B antigens, at least as measured by these procedures. Pulse-chase experiments, in conjunction with the isolation of cell-surface HLA-A and -B antigens by adsorbing the monoclonal antibody W6/32 to intact cells, indicate that nonglycosylated molecules reach the cell surface at a rate indistinguishable from that of fully glycosylated molecules (although the absolute amount synthesized in the presence of tunicamycin was decreased). Thus glycosylation is also not required for membrane insertion of HLA antigens, nor for their subsequent transport to the cell surface.

Animals↗
PubMed1980

Molecular cloning of a human histocompatibility antigen cDNA fragment.

A clone (pHLA-1) containing HLA-specific cDNA was constructed by reverse transcription of partially purified HLA mRNA from the human lymphoblastoid cell line LKT. The identity of pHLA-1 was established by its ability to hybridize to HLA heavy chain mRNA and by nucleotide sequence analysis. The pHLA-1 cDNA insert (approximately 525 base pairs) corresponds to the COOH-terminal 46 amino acids of an HLA-A, -B, or -C antigen (15 residues from the hydrophobic region and the remainder from the COOH-termial hydrophilic region), together with a portion of the 3' untranslated region of the mRNA.

Base Sequence↗
PubMed1980

An evaluation of the significance of amino acid sequence homologies in human histocompatibility antigens (HLA-A and HLA-B) with immunoglobulins and other proteins, using relatively short sequences.

A computer search was carried out for homologies between HLA-A and HLA-B antigen sequences and the sequences of constant and variable regions of immunoglobulins and of all other sequenced proteins. Searches were made both with relatively short peptide sequences from the HLA antigens and with those longer peptide sequences which were available in 1978. Significant homology of HLA antigen sequences to immunoglobulin constant region sequences was found in two cases: (1) a short decapeptide sequence which includes the fourth cysteine residue of HLA-B7 and (2) an 89-amino-acid residue (Ac-2) C-terminal fragment of the papainsolubilized HLA-B7 molecule. The difficulty of establishing statistically significant sequence homology with relatively short peptide sequences is emphasized by computer-based comparisons of the decapeptide sequence with randomly generated peptide sequences. It is concluded that statistically significant homology with short sequences can be assured only when extraordinarily high degrees of homology are present and additional constraints are included in the matches, for example, matches at relatively rare amino acid residues such as Cys, His and Trp. The homology of the 89-amino-acid residue sequence to constant region sequences of immunoglobulins is as great as or greater than that of beta 2-microglobulin. These findings and the unique domain structure involving a disulphide loop of comparable size strongly favour a common evolutionary origin for this region of HLA-A and -B, beta 2-microglobulin and immunoglobulin constant regions.

Amino Acid Sequence↗
PubMed1979

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen HLA-B7. 1. Isolation and amino acid composition of fragments and of tryptic and chymotryptic peptides.

As a part of the overall strategy for determining the complete covalent structure of the papain-solubilized portion of the heavy chain of the human histocompatibility antigen HLA-B7, the protein was dissected into various fragments by a combination of partial acid hydrolysis and cyanogen bromide cleavage. After purification by chromatographic procedures, these fragments have been used as a source for tryptic and chymotryptic peptides. Thirty-three major tryptic and twenty-two major chymotryptic peptides were purified in nanomole amounts and their amino acid compositions determined. These peptides account for the whole extent of the polypeptide chain with the exception of the amino-terminal CNBr pentapeptide. They provide the basis for the formal alignment of the acid cleavage and cyanogen bromide fragments of the molecule as well as the source material for the elucidation of the primary structure of the HLA-B7 heavy chain.

Amino Acid Sequence↗
PubMed1979

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen, HLA-B7. 2. Sequence determination and search for homologies.

The complete amino acid sequence of the papain-solubilized heavy chain of a human histocompatibility antigen (HLA-B7) has been elucidated. It consists of a polypeptide of 271 residues (31 333 daltons). A single glycan moiety is attached to an asparagine residue at position 86 by an N-glycosidic bond. Two intrachain disulfide bonds, arranged linearly, involve half-cystine residues at positions 101 and 164 and at positions 203 and 259. They form two loops of 62 and 55 residues, respectively, separated by 38 residues. Computer analysis of the sequence suggests the existence of internal homology between the amino-terminal portion (residues 1--90) and the region of the first disulfide loop (residues 91--180). There is a significant homology between the second disulfide loop region of the chain (residues 182-271) and immunoglobulin (Ig) constant domains and beta 2-microglobulin [Orr, H. T., Lancet, D., Robb, R. J., López de Castro, J. A., & Strominger, J. L. (1979A) Nature (London) (in press)]. However, no such homology to Ig is apparent in the amino-terminal or in the first disulfide loop regions.

Amino Acid Sequence↗
PubMed1979

The heavy chain of human histocompatibility antigen HLA-B7 contains an immunoglobulin-like region.

The 88-residue fragment (ac-2) containing the second disulphide loop from HLA-B7 heavy chain is shown to have statistically significant homology with Ig constant domains, including both matches at invariant positions and conservative substitutions of structurally important residues. Thus, both the light chain (beta 2m) and a segment of the heavy chain of HLA antigens may be structurally and evolutionarily related to immunoglobulins.

Amino Acid Sequence↗
PubMed1979

Assembly and maturation of HLA-A and HLA-B antigens in vivo.

HLA-A and HLA-B antigens are integral membrane glycoproteins which consist of a glycosylated heavy chain embedded in the membrane in noncovalent association with beta 2-microglobulin, a water-soluble polypeptide. The assembly and maturation of these antigens has been studied in vivo in the human B lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, -B27). Two antigenically distinct populations of HLA-A and -B heavy chains can be detected by antisera which recognize determinants sensitive to the conformation of the heavy chain. One heavy chain population is associated with beta 2-microglobulin, whereas the other population is not. These populations can be further distinguished by their oligosaccharide structure and their localization within the cell. Pulse-chase experiments demonstrate a precursor-product relationship between these heavy chain populations and suggest the following pathway for the assembly and maturation of HLA-A and -B antigens. The completed heavy chains initially carry high mannose oligosaccharides and are largely or wholly associated with beta 2-microglobulin. During the next 10-15 min, association with beta 2-microglobulin occurs and the heavy chain conformation is altered. Beginning at about 30 min after synthesis, the oligosaccharides are converted from the high mannose form to the complex form, and between 60 and 80 min after synthesis, the mature antigens appear at the cell surface. These observations are discussed in relation to in vivo and in vitro studies on the biosynthesis of a variety of secreted proteins and membrane proteins.

Cell Line↗
PubMed1979

Comparison of amino acid sequences of two human histocompatibility antigens, HLA-A2 and HLA-B7: location of putative alloantigenic sites.

The complete amino acid sequence for papain-solubilized HLA-B7 heavy chain is compared with the partial sequences of HLA-A2 and H-2Kb heavy chains. Although these molecules are highly conserved (i.e., 80% homology in comparing HLA-B7 with HLA-A2; 72% and 74% homology in comparing H-2Kb with HLA-A2 and HLA-B7, respectively), two stretches of greater variability are observed. These clusters of variability are discussed in terms of their possible involvement in the alloantigenic determinant(s) characteristic of these highly polymorphic membrane antigens.

Amino Acid Sequence↗