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J Trowsdale

Publications and source records attributed to J Trowsdale.

At least 55 records · Page 3Linked to original sources
PubMed2000

The human major histocompatability complex: lessons from the DNA sequence.

The entire 3.6-MbpDNA sequence of a human major histocompatibility complex derived from a composite of DNA clones from different haplotypes, was completed in 1999, primarily through the work of four main groups. At that time, it was the longest contiguous human DNA sequence to have been determined. The sequence is of extremely high quality and accuracy. In this review, we discuss how the DNA sequence has facilitated our understanding of the biology and genetics of the major histocompatibility complex. We suggest some ways in which the sequence may be exploited in the future to explore the relationship between the extraordinary polymorphism of the region and its association with both autoimmune and infectious diseases.

Animals↗
PubMed2000

Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei.

The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.

Cell Line↗
PubMed2000

DAP12 and KAP10 (DAP10)-novel transmembrane adapter proteins of the CD3zeta family.

Transmembrane adapter proteins are molecules that associate with receptors and mediate intracellular signals following interaction of the receptor with its ligand. Many such molecules have been characterized in detail, particularly the small TM adapters of the CD3zeta class at the core of the T cell receptor. Recently, two new genetically linked members of this class of transmembrane adapters have been identified called DAP12 (KARAP) and KAP10 (DAP10), respectively. In this review, we discuss this new class of TM adapters using the wealth of knowledge concerning CD3zeta and FcRgamma to highlight similarities and differences with DAP12 and KAP10. In addition, novel receptor families which interact with these TM adapters have also been identified. The role of these receptors and their inhibitory isoforms are discussed.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Generation of an immunodominant CTL epitope is affected by proteasome subunit composition and stability of the antigenic protein.

Generation of the HLA-A0201 (A2) influenza Matrix 58-66 epitope contained within the full-length Matrix protein is impaired in cells lacking the proteasome subunits low molecular protein 2 (LMP2) and LMP7. This Ag presentation block can be relieved by transfecting the wild-type LMP7 cDNA into LMP7-deficient cells. A mutated form of LMP7, lacking the two threonines at the catalytic active site, was equally capable of relieving the block in presentation of the influenza Matrix A2 epitope. These observations were extended by analyzing whether modification of the influenza Matrix protein could overcome the block in presentation of the A2 Matrix epitope. Expression of either a rapidly degraded form of the full-length Matrix protein or shorter Matrix fragments led to an efficient presentation of the A2 influenza Matrix epitope by LMP7-negative cells. These findings demonstrate two main points: 1) LMP7 incorporation into the proteasome is of greater importance for the generation of the influenza A2 Matrix epitope than the presence of the LMP7's catalytic site; and 2) the interplay between cytosolic proteases and stability of target proteins is of importance in optimization of Ag presentation. These observations may have relevance to the immunodominance of tumor and viral epitopes and raise the possibility that generation of shorter protein fragments could be a mechanism to ensure optimal Ag presentation by cells expressing low levels of LMP7.

Antigen Presentation↗
PubMed1999

Antigen presentation: TAP dances with ATP.

Assembly of antigen-presenting complexes between class I MHC molecules and peptide requires formation of a complex between the 'ABC' peptide transporter, TAP, and newly synthesized class I molecules. Recent studies have provided new insights into the role of ATP in peptide binding, transport and release.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Cutting edge: KAP10, a novel transmembrane adapter protein genetically linked to DAP12 but with unique signaling properties.

Transmembrane adapter proteins are a class of molecules that mediate signals from an extracellular receptor to the cytoplasm of the cell. We have cloned a novel transmembrane adapter protein called KAP10, a approximately 10-kDa protein that is encoded within 100 bp of the DAP12 locus on human chromosome 19. KAP10 is predominantly expressed in immune cells, including NK cells, T cells, and monocytes. We show that KAP10, unlike other transmembrane adapter proteins, binds phosphatidylinositol-3 kinase following phosphorylation of a cytoplasmic YINM motif, which results in activation of Akt. In addition, we identify KAP10 as being able to bind the adapter protein Grb2. Based on our data, we suggest that this molecule is involved in stimulation and costimulation in cells of both myeloid and lymphoid origin.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Gene organisation, sequence variation and isochore structure at the centromeric boundary of the human MHC.

We have mapped and sequenced the region immediately centromeric of the human major histocompatibility complex (MHC). A cluster of 13 genes/pseudogenes was identified in a 175 kb PAC linking the TAPASIN locus with the class II region. It includes two novel human genes (BING4 and SACM2L) and a thus far unnoticed human leucocyte antigen (HLA) class II pseudogene, termed HLA-DPA3. Analysis of the G+C content revealed an isochore boundary which, together with the previously reported telomeric boundary, defines the MHC class II region as one of the first completely sequenced isochores in the human genome. Comparison of the sequence with limited sequence from other cell lines shows that the high sequence variation found within the classical class II region extends beyond the identified isochore boundary leading us to propose the concept of an "extended MHC". By comparative analysis, we have precisely identified the mouse/human synteny breakpoint at the centromeric end of the extended MHC class II region between the genes HSET and PHF1.

Amino Acid Sequence↗
PubMed1999

IFN-gamma exposes a cryptic cytotoxic T lymphocyte epitope in HIV-1 reverse transcriptase.

The proteasome, an essential component of the ATP-dependent proteolytic pathway in eukaryotic cells, is responsible for the degradation of most cellular proteins and is believed to be the main source of MHC class I-restricted antigenic peptides for presentation to CTL. Inhibition of the proteasome by lactacystin or various peptide aldehydes can result in defective Ag presentation, and the pivotal role of the proteasome in Ag processing has become generally accepted. However, recent reports have challenged this observation. Here we examine the processing requirements of two HLA A*0201-restricted epitopes from HIV-1 reverse transcriptase and find that they are produced by different degradation pathways. Presentation of the C-terminal ILKEPVHGV epitope is impaired in ME275 melanoma cells by treatment with lactacystin, and is independent of expression of the IFN-gamma-inducible proteasome beta subunits LMP2 and LMP7. In contrast, both lactacystin treatment and expression of LMP7 induce the presentation of the N-terminal VIYQYMDDL epitope. Consistent with these observations we show that up-regulation of LMP7 by IFN-gamma enhances presentation of the VIYQYMDDL epitope. Hence interplay between constitutive and IFN-gamma-inducible beta-subunits of the proteasome can qualitatively influence Ag presentation. These observations may have relevance to the patterns of immunodominance during the natural course of viral infection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Isolation, sequencing and expression of RED, a novel human gene encoding an acidic-basic dipeptide repeat.

A novel human gene RED, and the murine homologue, MuRED, were cloned. These genes were named after the extensive stretch of alternating arginine (R) and glutamic acid (E) or aspartic acid (D) residues that they contain. We term this the 'RED' repeat. The genes of both species were expressed in a wide range of tissues and we have mapped the human gene to chromosome 5q22-24. MuRED and RED shared 98% sequence identity at the amino acid level. The open reading frame of both genes encodes a 557 amino acid protein. RED fused to a fluorescent tag was expressed in nuclei of transfected cells and localised to nuclear dots. Co-localisation studies showed that these nuclear dots did not contain either PML or Coilin, which are commonly found in the POD or coiled body nuclear compartments. Deletion of the amino terminal 265 amino acids resulted in a failure to sort efficiently to the nucleus, though nuclear dots were formed. Deletion of a further 50 amino acids from the amino terminus generates a protein that can sort to the nucleus but is unable to generate nuclear dots. Neither construct localised to the nucleolus. The characteristics of RED and its nuclear localisation implicate it as a regulatory protein, possibly involved in transcription.

Animals↗
PubMed1999

Sodium dodecyl sulfate-resistant HLA-DR "superdimer" bands are in some cases class II heterodimers bound to antibody.

The detection of dimers of dimers in MHC class II crystals has excited speculation about their possible functions in T cell Ag recognition. Biochemical evidence for the existence of DR superdimers falls short of proof and is controversial. To monitor B lymphoma cells for high m.w. complexes of HLA-DR molecules, membrane preparations and cell lysates were screened by one- and two-dimensional Western blotting. Under these conditions, in which DRalpha beta heterodimers were readily detected, no DR complexes with an (alpha beta)2-chain composition could be identified. Two mAbs (L243 and D1-12) immunoprecipitated high m.w. DR complexes suspected to be superdimers. However, biochemical analysis revealed that, rather than superdimers, these were SDS-stable complexes of DR in combination with the Abs. Thus, previous observations of HLA-DR superdimer bands may also reflect complexes of DR molecules with bound Ab.

Antibodies, Monoclonal↗
PubMed1999

Detection of polymorphism in the RING3 gene by high-throughput fluorescent SSCP analysis.

We describe the use of a high-throughput, fluorescent, polymorphism-detection system, based on single-strand conformation polymorphism to screen for polymorphism in the RING3 gene. This is the first extensive mutation screen of this major histocompatibility complex-linked gene, and the entire coding region and intron-exon junctions were examined by multiplexing over 3000 polymerase chain reaction products. These techniques should be applicable for analysis of variation in other human genes. Investigation of DNA from acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) patients, as well as healthy individuals revealed low levels of polymorphism across the RING3 gene. Comparison of the distribution of genotypes at each polymorphic site between patients and healthy individuals revealed a single site which significantly deviates from Hardy-Weinberg proportions.

Fluorescent Dyes↗
PubMed1999

Organization of the genes encoding the human proteasome activators PA28alpha and beta.

Two proteasome activators PA28alpha and beta, which have been implicated in antigen processing for loading class I MHC molecules, are synthesized in response to Ifn-gamma. The human genes encoding these activators (PSME1 and PSME2, respectively) were analyzed by sequencing. Each gene comprised 11 exons, consistent with gene duplication during vertebrate evolution. The intron/exon organization of both genes was highly conserved, the major difference being the absence of the exon encoding the lysine and glutamic acid-rich 'KEKE' motif in PA28beta. Two other genes of relevance to the immune system were located close to those for PA28 at 14q11.2 including ISGF3G, a protein involved in transcription after IFNalpha signalling. These sequences were also characterized.

Amino Acid Sequence↗
PubMed1999

Cloning of a new lectin-like receptor expressed on human NK cells.

Natural killer (NK) cells constitute the third major population of lymphocytes. They possess the inherent capacity to kill various tumor and virally infected cells and mediate the rejection of bone-marrow grafts in lethally irradiated animals. A large family of NK cell receptors belong to the C-type lectin superfamily and are localized to the NK gene complex on Chromosome (Chr) 6 in the mouse and Chr 12 in the human. Genes in the NK gene complex encode type II receptors and examples include the families of NKR-P1, Ly-49, and NKG2 receptors. Examples of other C-type lectin-like NK cell receptors that occur as individual genes are CD94, CD69, and AICL. Here we report the molecular characterization and chromosomal mapping of a human lectin-like transcript (LLT1) expressed on NK, T, and B cells and localized to the NK gene complex within 100 kilobases of CD69. The cDNA encodes a predicted protein of 191 amino acid residues with a transmembrane domain near the N-terminus and an extracellular domain of 132 amino acid residues with similarity to the carbohydrate recognition domain of C-type lectins. The predicted protein of LLT1 shows 59 and 56% similarity to AICL and CD69, respectively. The predicted protein does not contain any intracellular ITIM motifs, suggesting that LLT1 may be involved in mediating activation signals.

Amino Acid Sequence↗