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J L Strominger

Publications and source records attributed to J L Strominger.

At least 325 records · Page 18Linked to original sources
PubMed1985

Genetic complexity and expression of human class II histocompatibility antigens.

The genes encoding nearly all of the serologically defined class II antigens of the major histocompatibility complex have been isolated. Three class II loci have been studied in great detail. The DR region contains a single alpha gene and 3 beta chain genes, 1 of which is a pseudogene. The DR alpha chain gene has been linked to a DR beta gene which encodes a beta protein which contains the serological determinant MT3. A second cosmid cluster contains 2 beta genes, 1 of which encodes the DR4 allospecificity. The identification of these genes has been made by the comparison of amino terminal sequences of DR molecules obtained from a DR4 cell line and the deduced protein sequences of the beta 1 exons from cosmid and phage clones. A conserved element including the promoter and signal sequence is found at the 5' end of each of the 3 DR beta genes. Additionally, this element occurs three more times in the DR region, raising the question of whether additional beta chain genes might be found. The DQ region contains 2 pairs of genes, 1 of which encodes the DQ antigen. The 2nd pair of genes, called DX alpha and beta, appears to be capable of expressing a DQ-related product, although, to date, there is no evidence for its expression. The DP region also contains 2 pairs of genes. One pair encodes the DP antigen while the 2nd alpha-beta pair is shown to be composed of pseudogenes. The location of polymorphic regions in these genes and aspects of their relationship to the serology, evolution, and function of the class II MHC are discussed. The control of expression of class II genes by gamma-interferon has been examined. The promoters of class II genes are characterized by two conserved sequences common to all alpha and beta chain genes as well as by conserved sequences specific for either alpha or beta chain genes. In addition to studies of expression by DNA-mediated gene transformation, a system for the gene transfer of MHC antigens utilizing transmissible retrovirus vectors is described. Retrovirus vectors have been used to transmit DR alpha, DR beta, and the invariant chain (gamma) sequences to recipient cells with resultant expression of these proteins.

Alleles↗
PubMed1985

Structural analysis of the functional sites of class I HLA antigens.

Considerable knowledge of the molecular organization of class I HLA antigens has been attained through extensive structural analysis of these proteins and their genes. Particularly, the nature and location of the polymorphic regions has been established, as well as the basic patterns of structural variability. This work has not unveiled the functionally relevant sites of the HLA molecules but has provided the basis to develop new strategies to do so. The molecular analysis of the determinants recognized by specific antibodies and cytolytic T lymphocytes is being approached through the biochemical characterization of mutants induced in vitro and population variants that are selected by their loss of specific serological or CTL allodeterminants. Other approaches include the immunological analysis of sera raised against synthetic peptides whose structure mimics highly variable segments of class I HLA molecules. These studies have already revealed the participation of several regions in specific allorecognition by antibodies or CTLs and their potential is becoming increasingly evident. A new and possibly powerful approach is currently being used for the dissection of functional sites. It makes use of the structural information derived from sequence analysis and involves expression of cloned HLA genes in transfected mouse or human cells in conjunction with site-directed mutagenesis techniques. Although some difficulties still lie ahead in developing a system suitable for functional assays, the possibility of tailoring HLA mutants and studying the modulation of their recognition determinants by predetermined structural alterations open new pathways to the molecular analysis of HLA function.

Amino Acid Sequence↗
PubMed1985

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.

Acyltransferases↗
PubMed1985

Transformation by Epstein-Barr virus requires DNA sequences in the region of BamHI fragments Y and H.

Eight independent recombinant Epstein-Barr virus genomes, each of which was a transforming strain, were made by superinfecting cell lines containing Epstein-Barr virus DNA (Raji or B95-8 strain) with a nontransforming virus (P3HR1 strain). A knowledge of the constitution of each transforming recombinant allowed the localization of the defect in the genome of the nontransforming parent to a 12-megadalton sequence within the EcoRI A fragment. Within this region, the nontransforming virus has a deletion of the BamHI Y fragment and about half of the sequences in the adjacent BamHI H fragment. The present data suggest that this deletion is responsible for the nontransforming phenotype. Furthermore, mapping a deletion in one of the recombinant genomes allowed the conclusion that a sequence (comprising about 20% of the Epstein-Barr virus genome) from the center of BamHI-D to BamHI-I' is not necessary for the maintenance of transformation by Epstein-Barr virus.

Base Sequence↗
PubMed1985

Sequence analysis of the human major histocompatibility gene SX alpha.

The DP subregion of the human major histocompatibility complex contains two closely linked gene pairs, DP alpha, DP beta and SX alpha, SX beta. The exon-intron organization and the complete DNA sequence of the SX alpha gene are reported here. There are several mutations within the SX alpha gene which strongly suggest that it is a pseudogene. These include two frameshift mutations, one in the alpha 1 domain and the other in the cytoplasmic domain. A 5' splice site mutation at the end of the alpha 1 exon also exists. DNA sequence homology between DP alpha and SX alpha suggests that these genes arose through a gene duplication event.

Base Sequence↗
PubMed1985

Clonal T lymphocyte recognition of the fine structure of the HLA-A2 molecule.

A human alloimmune cytotoxic T lymphocyte (CTL) clone (4E4) was generated against the HLA-A2 molecule. Lysis of 51Cr-labeled HLA-A2 target cells was blocked by monoclonal antibodies (mAb), including mAb PA2.1 (anti-HLA-A2), mAb BB7.2 (anti-HLA-A2), mAb 4B (anti-HLA-A2-plus-A28), mAb MA2.1 (anti-HLA-A2-plus-B17), and mAb W6/32 (anti-HLA-A,B,C), which are directed against different serologic epitopes on the HLA-A2 molecule. However, HLA-A2 mutant lines lacking the serologic epitope recognized by mAb BB7.2 (anti-HLA-A2) were efficiently lysed by CTL 4E4. Thus, although mAb may block cytolysis, the HLA-A2 epitope recognized the 4E4 CTL clone is distinct from the HLA-A2-specific epitope recognized by serologic reagents. Moreover, analysis of HLA-A2 population variants revealed that only the predominant HLA-A2.1 subtype molecule was recognized by CTL 4E4. No cross-reactivity on other, biochemically related HLA-A2 population subtypes was observed, including HLA-A2.2 cells (Hill, CVE, ZYL, M7), HLA-A2.3 cells (TENJ, DK1), or HLA-A2.4 cells (CLA, KNE). This CTL clone appears to recognize a single epitope and, like monoclonal antibody counterparts, can be used to discriminate among immunogenic cellular and serologic epitopes on closely related HLA-A2 molecules. On the basis of the known sequence changes in mutant and subtype HLA-A2 molecules, it appears that the sequence spanning residues 147 to 157 may be critical for cellular recognition of this Class I MHC molecule.

Amino Acid Sequence↗
PubMed1984

Structure and polymorphism of the HLA class II SB light chain genes.

The HLA Class II region contains at least three groups of loci, DR, DC and SB, which play an important role in the immune response. The antigens encoded at these loci are heterodimers composed of an alpha and a beta chain. The sequence of a complete Class II beta cDNA clone whose sequence agrees closely with the limited N-terminal protein sequence available for the SB beta chain is reported. In addition the structure and coding sequence of genomic SB beta clones of two different SB haplotypes has been obtained and allows definition of some polymorphic regions. The SB beta gene appears to undergo alternate splicing at its 3' end, resulting in expression of two different intracytoplasmic regions. Partial sequencing of a second non-allelic SB beta-like gene, SX beta, indicates that it is a pseudogene.

Amino Acid Sequence↗
PubMed1984

HLA-A2 antigen phosphorylation in vitro by cyclic AMP-dependent protein kinase. Sites of phosphorylation and segmentation in class i major histocompatibility complex gene structure.

The heavy chain of the HLA-A2 antigen is phosphorylated by cyclic AMP-dependent protein kinase at two serine residues of the intracellular region. Limited proteolysis was performed on purified [32P]HLA-A2 antigens in order to define the sites of phosphorylation. Both of the phosphorylated serine residues are located in the carboxyl terminus of the heavy chain; one is encoded by exon 5, while the other is encoded by exon 6. The phosphoserine encoded by exon 5 is part of the conserved sequence Arg-Arg-Lys-Ser-Ser. This protein sequence contains the proper arrangement of amino acids for recognition and phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. In the murine class I antigens (H-2), exon 5 encodes a similar sequence of basic residues followed by one intervening residue and a threonine rather than a serine residue in the last amino acid position. A composite figure is presented that compares the carboxyl-terminal sequences of human and murine class I antigens and illustrates the known sites of phosphorylation recognized by various kinases. Each site of phosphorylation in the carboxyl terminus is contained within a conserved protein sequence encoded by one of the three exons. A separation and preservation of unique sites of phosphorylation could explain why there is segmentation in the genomic arrangement of class I molecules.

Amino Acid Sequence↗
PubMed1984

Identification of shared antigenic determinants of the putative human T lymphocyte antigen receptor.

A mouse antiserum, anti-gp40,49 was obtained by immunizing BALB/c mice with the putative T cell antigen receptor isolated from HPB-MLT cells. This antiserum reacted with peripheral blood lymphocyte (PBL) and a panel of immunocompetent T cell lines and clones in each case precipitating from lysates of cells labeled by surface iodination, a disulfide-linked dimer consisting of an alpha subunit Mr (46,000-49,000) and a beta subunit Mr (40,000-45,000). Variability in Mr of the two subunits, particularly of the beta (light) subunit, was observed when the receptors of immunocompetent T cell lines with different antigen specificities were compared. Anti-gp40,49 serum reacted selectively with the alpha subunit after reduction and alkylation of the protein complex. These results confirm the relationship between the gp40,49 protein complex of HPB-MLT cells and the putative T cell antigen receptor on normal immunocompetent T cells and indicate that the alpha subunit of the human receptor expressed shared determinant(s) that are immunogenic in the mouse. Some features of the T cell antigen receptor appear to be unusual in that even with a xenoantiserum against the purified molecule, only antibodies against clonotypic determinants could be detected at the cell surface by quantitative immunofluorescence analysis.

Animals↗
PubMed1984

Human and murine class I MHC antigens share conserved serine 335, the site of HLA phosphorylation in vivo.

The site of phosphorylation of the HLA-B7 antigen in vivo is serine 335, which is located in the intracellular region. Pseudomonas fragi protease was used in limited proteolysis experiments of HLA antigens to identify the position of the phosphoserine residue. The intracellular region is composed of 30 amino acids at the carboxyl terminus of the heavy chain; up to nine serine residues are located in this region. Four of the serines are found at the distal end, and are encoded by exon 7 in both human and murine class I MHC antigens. Alignment of the protein and DNA sequences of the intracellular regions of human and murine class I antigens demonstrates conservation of the serine positions located within this exon. Realignment of exon 7, by introducing a gap in the murine sequences, increases homology across species, and reveals two conserved serines at 332 and 335 within the conserved sequence Ser-Asp/Glu-X-Ser(P)-Leu. The preservation of this sequence and the site of phosphorylation suggests that this modification of the intracellular region of histocompatibility antigens is functionally significant.

Amino Acid Sequence↗
PubMed1984

Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds.

Exogenous radioactive palmitic acid is incorporated post-translationally into the HLA-B and -DR heavy chains, but not HLA-A heavy chains or -DR light chains of the human B lymphoblastoid cells JY and T51 . Protease digestions localize the label to the transmembrane region of the B7 heavy chain. Both B7 and DR heavy chains have a cysteine in the transmembrane hydrophobic region, while the A2 heavy chain and DR light chains have none. Palmitic acid is covalently linked to these transmembrane cysteines via a thioester bond since: 1) the label is not removed by organic extraction or boiling in sodium dodecyl sulfate and dithiothreitol, but is released at room temperature by methanolic KOH as methyl palmitate, and by hydroxylamine as palmitohydroxamate . 2) The pH sensitivity and kinetics of release by hydroxylamine and Tris are similar to those of palmitoyl-CoA (thioester linkage) and unlike those of methyl palmitate and palmitoyllysophosphatidylcholine ( hydroxyester linkages). 3) Neutral hydroxylamine treatment (but not neutral Tris treatment) generates sites that can be reduced and alkylated in the transmembrane region of B7 heavy chain and to a lesser extent in DR heavy chain. 4) Organic extraction of pronase digests of labeled B7 yields peptides containing palmitate and cysteine (but not serine or threonine) which co-migrate by thin layer chromatography. A population of beta 2-microglobulin molecules not associated with heavy chains is palmitylated , but not via a thioester linkage.

Cysteine↗
PubMed1984

Genetic and biochemical characterization of human lymphocyte cell surface antigens. The A-1A5 and A-3A4 determinants.

The genes that code for the human lymphocyte cell surface determinants defined by monoclonal antibodies A- 1A5 and A- 3A4 have been genetically mapped. All human chromosomes, except Y, were included in a series of human less than mouse lymphocyte hybrid populations that retained expression of lymphocyte-specific surface markers. Expression of the A- 1A5 and A- 3A4 antigens was quantitated by indirect immunofluorescence and fluorescence-activated cell sorter (FACS) analysis. Hybrid populations heterogeneous for antigen expression were sorted to yield antigenically homogeneous subpopulations. Isozyme analysis indicated concordant segregation of the A- 1A5 determinant with chromosome 10, and the A- 3A4 determinant with chromosome 4. In contrast to the unhybridized human parent cell line (MOLT-4), from which A- 1A5 immunoprecipitated two proteins (160,000 and 125,000 Mr), A- 1A5 only immunoprecipitated a single band (125,000 Mr) from an A- 1A5 -expressing human less than mouse hybrid. The genetic disassociation of these two proteins from the A- 1A5 -reactive complex suggests that the appearance of the 160,000 Mr protein requires a gene locus that is unlinked to the locus for the 125,000 Mr protein on chromosome 10. A third component of the A- 1A5 -reactive protein complex (210,000 Mr), which is recognized by the monoclonal antibody TS2/7, was not expressed on the parent MOLT-4 cells, but was weakly expressed on MOLT-4 less than mouse BW5147 hybrids. This allowed preliminary mapping of that determinant to either chromosome 10 or 15. The A- 3A4 antigen (approximately 45,000 Mr) is a novel cell surface structure expressed on all hematopoietic cell lines tested, and represents the first cell surface marker mapped to chromosome 4.

Animals↗
PubMed1984

Synthesis of peptidoglycan by high molecular weight penicillin-binding proteins of Bacillus subtilis and Bacillus stearothermophilus.

The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems. In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper. The pH optimum for peptidoglycan synthesis was 7.5 for B. subtilis PBPs 1, 2, and 4 and 8.5 for B. stearothermophilus PBPs 1-4. Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents. Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur. Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin, and macarbomycin were strong inhibitors. In a test tube assay system, optimal synthesis occurred either in the presence of 10% ethylene glycol, 10% glycerol, and 8% methanol or in the presence of 10% N-acetylglucosamine. The products of lysozyme digestion of the synthesized peptidoglycan were analyzed by gel filtration and paper chromatography. B. stearothermophilus PBPs 1-4 synthesized a peptidoglycan product that was 5-7% cross-linked. No evidence for cross-linking was apparent in the peptidoglycan product of B. subtilis PBPs 1, 2, and 4.

Bacillus subtilis↗
PubMed1984

Purification and properties of penicillin-binding proteins 5 and 6 from the dacA mutant strain of Escherichia coli (JE 11191).

Penicillin-binding proteins 5 and 6 have been purified to homogeneity from the dacA mutant strain of Escherichia coli (JE 11191). Protein 6 from the mutant strain appears to be identical to that from the wild type, but protein 5 is a mutant protein which has no D-alanine carboxypeptidase activity. Moreover, the mutant protein 5 binds, but does not release, [14C]penicillin G. Correspondingly, an acyl-enzyme intermediate derived from a synthetic substrate is accumulated by the mutant protein. A comparison of the acylation site for the synthetic substrate and for penicillin G by limited proteolysis and some other properties of the mutant protein are described.

Acylation↗
PubMed1984

Inter-locus and intra-allelic polymorphisms of HLA class I antigen gene mRNA.

We have constructed cDNA clone libraries from two lymphoblastoid cell lines, JY (HLA-A2, B7, C untypeable) and LB (HLA-A28, B40, Cw3), and isolated clones encoding class I HLA antigens. We have characterized short oligonucleotide probes derived from the coding region of the HLA class I antigens which are specific for the HLA-A and -B loci. These probes have been used to subdivide the class I cDNA clones into subclasses. DNA sequencing of several HLA-A and -B related clones has allowed us to extend the primary structural characterization of these cell-surface antigens. This analysis has also detected a sequence polymorphism at the HLA-A locus, indicating that the previously considered homozygous typing cell line LB expresses two alleles of similar, although not identical, serological specificity.

Alleles↗
PubMed1984

Identification of three class II antigens, DR7, MB2, and MT3, from a homozygous human cell line.

The class II antigens from a homozygous DR7 human B cell line were isolated by immunoprecipitation with human alloantisera or monoclonal antibodies. Three class II light chains and three class II heavy chains were identified. The heavy and light chain subunits of the DR7 antigen were distinct from those of the MB2 antigen as assessed by two-dimensional gel electrophoresis. A third light chain in addition to the DR7 and MB2 subunits was precipitated by some alloantisera containing MT3 antibodies. The monoclonal antibodies examined immunoprecipitated one or more of the three class II antigens. The MB2 antigen appeared to be the same as the major antigen recognized by L227 and the DS7 antigen precipitated by SG171.

Antibodies, Monoclonal↗