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J Trowsdale

Publications and source records attributed to J Trowsdale.

At least 19 recordsLinked to original sources
PubMed1992

DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing.

The genomic sequence of a 66,109 bp long region within the human MHC has been determined by manual and automated DNA sequencing. From cDNA mapping and sequencing data it is known that this region contains a cluster of at least four genes that are believed to be involved in antigen processing. Here, we describe the genomic organization of these genes, which comprise two proteasome-related genes (LMP2 and LMP7), thought to be involved in the proteolytic degradation of cytoplasmic antigens and two ABC transporter genes (TAP1 and TAP2), thought to be involved in pumping of the degraded peptides across the endoplasmic reticulum membrane. Analysis of the sequence homology and the intron/exon structures of the corresponding genes suggests that one gene pair arose by duplication from the other. Comparison of the available sequence data from other organisms shows striking conservation (70 to 84%) of this gene cluster in human, mouse and rat. The presence of several potential interferon stimulated response elements (ISREs) is in agreement with the experimentally observed up-regulation of these genes with gamma-interferon.

Amino Acid Sequence↗
PubMed1992

An ovarian tumor marker with homology to vaccinia virus contains an IgV-like region and multiple transmembrane domains.

The monoclonal antibody OVTL3 has a highly restricted reactivity with ovarian carcinomas and defines a surface glycoprotein, OA3, which has been used for immunotargeting. To understand why OA3 is found on ovarian tumors we isolated a complementary DNA by expression cloning. The clone encodes a 323-amino acid protein with 5 putative membrane spanning domains, reminiscent of a membrane receptor or channel, but of a new type with little or no sequence similarity with these families of proteins. Interestingly, the OA3 sequence is highly related to a vaccinia virus encoded protein (VA38) and its extracellular domain is a member of the immunoglobulin V region superfamily.

Amino Acid Sequence↗
PubMed1992

Mitotic recombination of yeast artificial chromosomes.

Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.

Blotting, Southern↗
PubMed1992

Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi.

Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Polymorphism in a second ABC transporter gene located within the class II region of the human major histocompatibility complex.

Recent studies have identified genes within the major histocompatibility complex (MHC) that may play a role in presentation of antigenic peptides to T cells. We have previously described RING4, a gene within the human MHC class II region that has sequence homology with members of the ABC ("ATP-binding cassette") transporter superfamily. We now report the nucleotide sequence of RING11, a second ABC transporter gene located approximately 7 kilobases telomeric to RING4, RING11 is gamma-interferon inducible, a property shared with other genes involved in antigen presentation. Comparison between the amino acid sequences of RING11 and RING4 reveals strong homology. We propose that they form a heterodimer that transports peptides from the cytoplasm into the endoplasmic reticulum. We have identified two RING11 alleles, which differ in the length of their derived protein sequence by 17 amino acids. The more common of these alleles is present in a Caucasoid population at a frequency of 79%.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex.

Presentation of cytoplasmic antigens to class I-restricted cytotoxic T cells implied the existence of a specialized peptide transporter. For most class I heavy chains, association with peptides of the appropriate length is required for stable assembly with beta 2-microglobulin. Mutant cells RMA-S and .174/T2 neither assemble stable class I molecules nor present intracellular antigens, and we have suggested that they have lost a function required for the transport of short peptides from the cytosol to the endoplasmic reticulum. The genetic defect in .174 has been localized to a large deletion in the class II region of the major histocompatibility complex, within which two genes (RING4 and RING11) have been identified that code for 'ABC' (ATP-binding cassette) transporters. We report here that the protein products of these two genes assemble to form a complex. Defects in either protein result in the formation of unstable class I molecules and loss of presentation of intracellular antigens. The molecular defect in a new mutant, BM36.1, is shown to be in the ATP-binding domain of the RING11/PSF2 protein. This is in contrast to the mutant .134, which lacks the RING4/PSF1 protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Assignment of the human homologue of the mouse t-complex gene TCTE3 to human chromosome 6q27.

The gene TCTE3 from the mouse t-complex region is expressed specifically in testicular germ cells. It maps in the central subregion of the t-complex on mouse chromosome 17 containing loci involved in transmission ratio distortion and male sterility. In this study, somatic cell hybrid lines have been used to map the human homologue, TCTE3, to the long arm of chromosome 6. CISS hybridization with the human lambda clone h117 refined this chromosome assignment to the very distal position of chromosome 6q27, thus providing further evidence that loci from the t-complex of mouse chromosome 17 can map to opposite arms of human chromosome 6.

Animals↗
PubMed1992

A testis-expressed Zn finger gene (ZNF76) in human 6p21.3 centromeric to the MHC is closely linked to the human homolog of the t-complex gene tcp-11.

A novel testis-expressed Zn finger gene (ZNF76) was identified by screening cDNA libraries with cosmids derived from 6p21. ZNF76 is a member of the GLI-Krüppel family of DNA binding proteins. It is conserved in mouse where transcription in testis is initiated at Day 20 after birth. The mouse tcp-11 gene is located in the distal inversion of the t-complex and is developmentally regulated in the same manner as ZNF76. The human homolog of tcp-11 was isolated to allow a precise chromosomal localization. By using a combination of somatic cell hybrids, radiation hybrids, metaphase and interphase fluorescent in situ hybridization, and pulsed-field gel electrophoresis, we mapped the two genes to the 6p21.2 to 6p21.3 region and linked them to each other within 300 kb of DNA, approximately 2 Mb centromeric to the major histocompatibility complex.

Amino Acid Sequence↗
PubMed1992

Genomic organization of the human folate receptor genes on chromosome 11q13.

There has been interest in the high affinity folate receptor (FOLR) recently because of its high expression in the majority of ovarian tumors. The FOLR genes are part of a family that includes an adult gene, a fetal gene, and one or more pseudogenes, which have been localized to chromosome 11. As a step toward understanding why the adult FOLR gene product is expressed on tumors, we have determined the organization of all the human FOLR-related genes. YAC clones were isolated using the adult FOLR probe. The organization of the locus was determined by PFGE of YAC DNA and by YAC fragmentation. Four FOLR-related genes were found within 140 kb. The adult and fetal genes are not more than 23 kb apart, with the 3' end of the adult gene facing the 5' of the fetal gene. A physical map of over 900 kb of the surrounding region was also constructed. The chromosomal assignment of the FOLR locus was refined to 11q13.3-q13.5 telomeric of the FGF3 locus using fluorescence in situ hybridization.

Adult↗
PubMed1992

The MHC: relationship between linkage and function.

It is intriguing that several genes with associated functions, including all of class I and class II genes, as well as some genes affecting antigen presentation of both class I and class II pathways, are linked in the MHC. Recent observations have led to speculation that there may be a functional explanation for keeping these related genes together.

Animals↗
PubMed1992

The LFA-3 adhesion pathway is differently utilized by superantigen-activated human CD4+ T-cell subsets.

The superantigen SEA binds to MHC class II molecules and activates a large fraction of T cells as a result of interaction with particular TCR-V beta sequences. MHC class II transfected CHO cells induce a marginal CD4+ T-cell proliferation in the presence of SEA. CHO cells transfected with both MHC class II and LFA-3 (HLA-DR4/LFA-3 double transfectants) supported a vigorous T-cell proliferation and required 1000-fold lower SEA concentration than DR4-transfected cells. DR4/LFA-3 double transfectants presenting SEA to CD4+ T cells induced large amounts of IFN-gamma, while single DR4 transfectants failed to elicit IFN-gamma production. CD4+45RA+ naive T cells proliferated much more strongly compared with CD4+45R0+ memory T cells when SEA was presented by the DR4/LFA-3-transfected cells. In contrast, IFN-gamma production was only detected in CD4+45R0+ memory cells. The enhanced proliferation by the CD4+45RA+ naive T cells was not due to a stronger binding to the accessory DR4/LFA-3 cells. Human CD4+ T-cell lines mediated a low level of SEA-dependent cell-mediated cytotoxicity (SDCC) against DR4 target cells, whereas a strong SDCC was mediated against DR4/LFA-3-expressing target cells. These results demonstrate that superantigen-activated human CD4+ T cells require the adhesion molecule LFA-3 for optimal stimulation and that the CD4+ naive and memory T-helper cells are different in their response to LFA-3 as an accessory molecule.

Animals↗
PubMed1992

A homologue of the Drosophila female sterile homeotic (fsh) gene in the class II region of the human MHC.

The RING3 gene maps in the class II region of the human major histocompatibility complex, at a CpG island distal of the HLA-DNA gene. RING3 cDNAs were obtained from a T cell cDNA library and the longest (4 kb) was sequenced. The sequence contained an open reading frame encoding a protein of 754 amino acids. A screen of protein databases revealed striking homology between the RING3 protein and the Drosophila female sterile homeotic gene (fsh) which is implicated in the establishment of segments in the early embryo. Partial sequence homology was also observed with some other proteins involved in cell cycle control (CCG1), cell division (ftsA) and regulation of cell growth (gamma interferons). This highly conserved gene may play an important role in human development. In addition, its location in the MHC class II region may be related to some HLA-associated diseases.

Amino Acid Sequence↗
PubMed1991

Second proteasome-related gene in the human MHC class II region.

Antgen processing involves the generation of peptides from cytosolic proteins and their transport into the endoplasmic reticulum where they associate with major histocompatibility complex (MHC) class I molecules. Two genes have been identified in the MHC class II region, RING4 and RING11 in humans, which are believed to encode the peptide transport proteins. Attention is now focused on how the transporters are provided with peptides. The proteasome, a large complex of subunits with multiple proteolytic activities, is a candidate for this function. Recently we reported a proteasome-related sequence, RING10, mapping between the transporter genes. Here we describe a second human proteasome-like gene, RING12, immediately centromeric of the RING4 locus. Therefore RING12, 4, 10 and 11 form a tightly linked cluster of interferon-inducible genes within the MHC with an essential role in antigen processing.

Amino Acid Sequence↗
PubMed1991

A new human HLA class II-related locus, DM.

HLA class II molecules have a crucial role in the immune response to antigens. We have isolated two new class II-like complementary DNA sequences, RING6 and RING7, which map between the HLA-DNA and -DOB loci. They are novel members of the immunoglobulin gene family which may have diverged before the duplications that gave rise to the main class II loci. The RING6 and RING7 genes seem to encode alpha- and beta-chains of a previously undiscovered class II-related protein.

Amino Acid Sequence↗
PubMed1991

Folate-binding protein is a marker for ovarian cancer.

We describe the isolation of a complementary DNA (cDNA) sequence encoding the ovarian cancer-associated antigen recognized by monoclonal antibody MOv18 and its identification as a high-affinity folate-binding protein (FBP). Functional cDNA clones were isolated using mRNA from the ovarian carcinoma cell line SKOV3 and colon carcinoma cell line HT29, by transient expression in WOP cells and selection of expressing cells by adhesion to antibody-coated magnetic beads. The cDNAs differed in the lengths of 5'- and 3'-noncoding regions, but they encoded identical peptides. A database search clearly showed them to be adult high-affinity FBPs with amino acid sequences identical with those isolated from normal placenta and several carcinoma cell lines. Reactivity of cell lines with MOv18 was quantitatively consistent with the expression of FBP mRNA. Southern hybridizations show evidence of a family of related genes and/or pseudogenes and were mapped to chromosome 11q13.3-14.1 by fluorescent in situ hybridization using cosmid clones containing part of this region. Also identified were two PstI polymorphisms of four and three alleles, respectively, and a two-allele MspI polymorphism. The folate-binding protein locus was not amplified in any of the 16 carcinoma cell lines tested and in only 1 of 10 serous adenocarcinomas, indicating that overexpression of FBP in ovarian cancer cannot, in general, be due to gene amplification.

Amino Acid Sequence↗
PubMed1991

A proteasome-related gene between the two ABC transporter loci in the class II region of the human MHC.

It is now possible to paint a detailed picture of how cytoplasmic proteins are handled by the immune system. They are apparently degraded in the cytoplasm into peptides. These are then transported into the endoplasmic reticulum where they encounter class I major histocompatibility complex (MHC) molecules. Once loaded with peptide, the HLA molecules move through the Golgi apparatus to the cell membrane. Until recently, it had not been established how peptides without signal sequences cross the ER membrane. However, a number of papers have now described a pair of membrane transporter genes of the ABC (ATP-binding cassette) super-family which are attractive candidates for this function. Both transporter genes, which may encode two halves of a heterodimer, are situated in the class II region of the MHC. There is evidence that other putative components of the processing machinery, the LMPs (low molecular mass polypeptides), are also encoded in the MHC. Similarities between the properties of the LMPs and a large intracellular protease complex, called proteasome, have led to the suggestion that LMPs are involved in processing antigens. We have now identified a human gene with sequence homology to proteasome components. Remarkably, this gene maps between the two putative peptide transporter genes.

Amino Acid Sequence↗