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Biomedical subjects

E O Long

Publications and source records attributed to E O Long.

At least 109 records · Page 6Linked to original sources
PubMed1986

Cell surface expression of class II histocompatibility antigens occurs in the absence of the invariant chain.

The invariant chain is a glycoprotein transiently associated with the alpha and beta subunits of class II antigens of the major histocompatibility complex during their transport to the cell surface. An expression assay with cDNA clones transfected into simian COS cells was used to test whether the invariant chain is required for assembly and transport of human class II antigens. COS cells do not express detectable levels of RNA from the endogenous invariant chain gene. Cell surface expression of the DP, DQ, and DR antigens was observed in COS cells transfected with the respective alpha and beta chain cDNA clones. Analysis of RNA from the transfected cells showed that the human genes were transcribed in COS cells and that the endogenous simian class II and invariant chain genes were not induced. Cotransfections with an invariant chain cDNA clone did not alter the levels of class II antigens at the cell surface. Biosynthetic labeling and immunoprecipitation demonstrated that the invariant chain cDNA was expressed into a protein which associated with DR alpha and beta chains. Efficient expression of DR antigen in absence of invariant chain was also observed at the surface of a human fibroblast line stably transfected with DR alpha and beta cDNA. This study demonstrates that expression of all three human class II antigens can be achieved with cDNAs cloned in expression vectors. Furthermore, cell surface expression of class II major histocompatibility complex antigens can occur in absence of invariant chain. The postulated role of the invariant chain in class II antigen transport to the cell surface must be reevaluated. The invariant chain may rather be involved in functional properties of class II molecules such as antigen presentation.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1985

DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression.

The HLA-D region of the human major histocompatibility complex encodes the genes for the alpha and beta chains of the DP, DQ and DR class II antigens. A cDNA clone encoding a new class II beta chain (designated DO) was isolated from a library constructed from mRNA of a mutant B-cell line having a single HLA haplotype. Complete cDNA clones encoding the four isotypic beta chains of the DR1, DQw1, DPw2 and putative DO antigens were sequenced. The DO beta gene was mapped in the D region by hybridization with DNA of HLA-deletion mutants. DO beta mRNA expression is low in B-cell lines but remains in mutant lines which have lost expression of other class II genes. Unlike other class II genes DO beta is not induced by gamma-interferon in fibroblast lines. The DO beta gene is distinct from the DP beta, DQ beta and DR beta genes in its pattern of nucleotide divergence. The independent evolution and expression of DO beta suggest that it may be part of a functionally distinct class II molecule.

Amino Acid Sequence↗
PubMed1984

Recombination within the HLA-D region. Correlation of molecular genotyping with functional data.

Molecular genotyping of the HLA-D/DR region in a family correlated with serologic and cellular typing data. It was further possible to predict a subtle difference in SB region-related functions from such molecular studies. A family that included an individual who inherited an HLA haplotype with a paternal recombination between HLA-B and the HLA-D/DR region was identified by classic HLA typing techniques. Segregation of HLA-D/DR region genes in this family was studied by Southern blot analysis using cDNA probes for DR alpha, DR beta, DC alpha, DC beta, and SB beta. Restriction enzyme fragment polymorphisms observed for every gene tested were in concordance with assigned HLA haplotypes (including the individual known to have inherited a paternal recombinant haplotype) with one exception: two HLA identical siblings were observed to have different SB beta restriction fragment patterns. Further testing revealed that one individual inherited a maternal HLA haplotype recombinant between the HLA-D/DR region and SB beta. Although both maternal SB alleles typed as SB4, allelic differences could be detected cellularly by primed lymphocytes and by the differential expression of a class II cell surface antigen using monoclonal antibody. Therefore, predicted and nonpredicted recombinant haplotypes were detected in a family by molecular genotyping.

DNA Restriction Enzymes↗
PubMed1984

The complete sequence of the mRNA for the HLA-DR-associated invariant chain reveals a polypeptide with an unusual transmembrane polarity.

A non-polymorphic polypeptide is associated intracellularly with the alpha and beta chains of murine Ia antigens and of human HLA-DR antigens. The exact role and the structure of this invariant chain have not been determined so far. A cDNA clone encoding the 33 000 dalton human invariant chain has been isolated. The nucleotide sequence of a near full-length cDNA clone, together with the sequence of the 5' portion of the mRNA determined by primer-extension, are reported here. The protein structure deduced from that sequence shows an unusual feature: the presence of a hydrophobic transmembrane region near the NH2 terminus, and of two glycosylation sites near the middle, indicates that the invariant chain has a polarity of membrane insertion which is inverted relative to histocompatibility antigens and most transmembrane proteins.

Amino Acid Sequence↗
PubMed1983

Complete sequence of an HLA-dR beta chain deduced from a cDNA clone and identification of multiple non-allelic DR beta chain genes.

At least three polymorphic class II antigens are encoded in the human major histocompatibility complex (HLA): DR, DC and SB. cDNA clones encoding beta chains of HLA-DR antigen, derived from mRNA of a heterozygous B-cell line, were isolated and could be divided into four subsets, clearly distinct from cDNA clones encoding DC beta chains. Therefore, at least two non-allelic DR beta chain genes exist. The complete sequence of one of the DR beta chain cDNA clones is presented. It defines a putative signal sequence, two extracellular domains, a trans-membrane region and a cytoplasmic tail. Comparison with a DC beta chain cDNA clone revealed a homology of 70% between the two beta chains and that the two genes diverged under relatively little selective pressure. A set of amino acids conserved in immunoglobulin molecules was found to be identical in both DR and DC beta chains. Comparison of the DR beta chain sequence with the amino acid sequence of another DR beta chain revealed a homology of 87% and that most differences are single amino acid substitutions. Allelic polymorphism in DR beta chains has probably not arisen by changes in long blocks of sequence.

Alleles↗
PubMed1983

Molecular analysis of the genes for human class II antigens of the major histocompatibility complex.

Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.

Alleles↗
PubMed1983

Isolation of cDNA clones for the p33 invariant chain associated with HLA-DR antigens.

HLA-DR antigens are polymorphic cell surface glycoproteins involved in the control of the immune response in man. They consist of two subunits, the alpha and the beta chains. In addition, an invariant glycoprotein of Mr 33,000 (DRp33) is associated intracellularly with HLA-DR antigens. A cDNA clone for DRp33, called 33-10, was isolated. Because no amino acid sequence has yet been determined for DRp33 the identification of cDNA clone 33-10 was based on selection of mRNA by hybridization, subsequent translation in a rabbit reticulocyte lysate supplemented with microsomes, and translation in microinjected Xenopus oocytes followed by immunoprecipitation with an anti-DR antiserum. The translation products assembled with DR alpha and beta chains in oocytes coinjected with all three mRNAs. Assembly of DR alpha and beta chains was also observed in the absence of DRp33 mRNA. Furthermore, when compared with DRp33 immunoprecipitated from a human B-cell line, translation products of the hybrid-selected mRNA showed (i) identical migration in two-dimensional gel electrophoresis, (ii) identical apparent molecular weight in the absence of N-linked glycosylation, and (iii) a very similar two-dimensional peptide map. Transcription of the DRp33 gene into a mRNA 1,400 nucleotides long was observed in B cells but was undetectable in T-cell lines and was very low in liver. Thus, DRp33 appears to be coordinately expressed with DR alpha and beta chains. Hybridization to DNA of mouse-human somatic cell hybrids showed that DRp33 is encoded by a gene that is located outside the major histocompatibility complex.

Animals↗
PubMed1982

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.

Animals↗
PubMed1982

Isolation of cDNA clones encoding HLA-DR alpha chains.

HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.

Animals↗
PubMed1982

Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.

cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines.

Base Sequence↗
PubMed1981

Expression of the ribosomal DNA insertions in bobbed mutants of Drosophila melanogaster.

We have shown earlier that interrupted rRNA genes in Drosophila melanogaster do not contribute significantly to rRNA production by a splicing mechanism (Long and Dawid 1979). In the work reported here the expression of interrupted rRNA genes was tested in several stocks that carry bobbed mutations, i.e., have partial deletions of their rRNA gene clusters. Transcripts of the major 5 kb type 1 insertion are very rare in bobbed flies as they are in the wild type, occurring at a concentration in embryos of less than one copy per nucleus. Transcripts of short type 1 insertions are more abundant in certain bobbed stocks, especially those carrying the car bb chromosome. However, other severely bobbed flies have no increase in these insertion transcripts over the wild-type levels. Type 2 insertions are transcribed into very rare RNA molecules in the wild type and in the bobbed genotypes that were studied. From these results we conclude that interrupted rRNA genes are not expressed through a splicing mechanism into mature rRNA in mutant or wild-type flies. Since even severely bobbed flies fail to utilize their interrupted rRNA genes, we suggest that these genes cannot be transcribed productively in D. melanogaster.

Animals↗
PubMed1981

Ribosomal insertion-like elements in Drosophila melanogaster are interspersed with mobile sequences.

Sequences homologous to the type 1 ribosomal DNA insertion occur in the chromocenter and in one location on the fourth chromosome of Drosophila melanogaster. Ribosomal insertion-like elements frequently occur in the form of tandem repeats and are often interspersed with nonhomologous DNA. Sequences that interrupt of flank insertion-like elements are members of diverged repeated DNA families. Members of five of these interspersed sequence families are located at multiple euchromatic sites as well as in the chromocenter. The euchromatic sites differ greatly in the gt-1 and gt-X11 stocks of D. melanogaster, suggesting that these sequence families are mobile in the genome. No long inverted repeats were detected in any of these interspersed sequences. One sequence, called 101F, interrupts a ribosomal insertion-like element; the nucleotide sequences across the boundaries between 101F and ribosomal insertion have been determined. A stretch of 13 base pairs that is present once in the ribosomal insertion is repeated at the ends of the 101F sequence, suggesting a target site duplication. Within the 101F element, no extended direct or inverted repeat sequence exists. The interspersed repeated sequences studied are transcribed into rare, heterogeneous, poly(A)-lacking nuclear RNA molecules. In one case, we showed that both strands of a flanking sequence are transcribed, but to a different extent. With respect to structural features and the nature of their transcripts, the sequences described here are distinct from other known transposable elements in Drosophila.

Animals↗
PubMed1981

Nucleotide sequence of the initiation site for ribosomal RNA transcription in Drosophila melanogaster: comparison of genes with and without insertions.

The sequence of 470 nucleotides surrounding the initiation site for rRNA transcription in Drosophila melanogaster has been determined. The precise initiation site was determined first by measuring the DNA fragment protected by the rRNA precursor against digestion by the single-strand specific nuclease S1 and second by direct sequence determination of the first 13 nucleotides of the rRNA precursor. Because greater than 80% od rRNA precursor molecules have been shown previously to bear pppA or ppA 5' termini, we assume that they represent the primary transcription product. Short sequence homologies exist with the initiation regions for rRNA transcription of Xenopus laevis and Saccharomyces cerevisiae. We have determined the nucleotide sequence of the initiation region in four cloned ribosomal genes from D. melanogaster which are not interrupted and in four cloned ribosomal genes in which the 28S rRNA coding region is interrupted by a 5-kilobase type 1 insertion. Three uninterrupted genes and three interrupted genes have identical sequences in the entire analyzed region. The remaining two genes have a single identical base substitution at position -17. We have shown previously that interrupted ribosomal genes in D. melanogaster are not effectively transcribed. Because the nucleotide sequences of the region where transcription initiates are identical in genes with or without insertions, we postulate that the presence of the insertion itself may be responsible for the inactivity of the interrupted genes.

Animals↗