Search PubMed⌕ Search

Biomedical subjects

J Trowsdale

Publications and source records attributed to J Trowsdale.

At least 145 records · Page 8Linked to original sources
PubMed1993

Efficiency and specificity of gene isolation by exon amplification.

Exon amplification is an increasingly popular approach to the identification of transcribed sequences and will complement other strategies to isolate genes. We have used this system to amplify candidate exons from 32 cosmids, including 8 cosmids which span a well characterized 185-kb region of the human major histocompatibility class II region on Chromosome (Chr) 6. We have examined the efficiency, specificity, and reproducibility of the system in isolating exons from genes known to be present on particular cosmids and have determined the nature and frequency of artefact amplifications in routine cosmid screening. We were able to clone at least one exon from 88% (7/8) of all known genes tested (including exons which are differentially spliced) and obtained artefacts from 19% (6/32) of the cosmids tested. Such artefacts generally arise from the amplification of noncoding sequences flanked by regions with high homology to acceptor and donor splice junctions. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids which have different numbers of genes and gene structures and describe several approaches to the characterization of novel exons cloned in this study.

Amino Acid Sequence↗
PubMed1993

A cosmid library specific for human chromosome regions 6p21.3 and 6q27.

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.

Base Sequence↗
PubMed1993

New vector for transfer of yeast artificial chromosomes to mammalian cells.

A modification vector has been constructed to facilitate the transfer of yeast artificial chromosomes (YACs) to mammalian cells in culture by targeting a dominant selectable marker (G418 resistance) to the right arm of pYAC4 clones. The ADE2 gene is used for yeast selection with consequent disruption of the URA3 gene, allowing direct modification of YACs within the common host strain AB1380, and providing a simple test for correct targeting. This vector has been tested by modification of a 550-kb YAC containing part of the human MHC class II region and transfer to CHO cells by protoplast fusion. Analysis of 15 independent G418-resistant CHO lines obtained following fusion suggests the majority contain a complete YAC with moderate amplification in some lines.

Animals↗
PubMed1993

Genomic structure and function in the MHC.

The major histocompatibility complex (MHC) is a fascinating region of the human genome. More is known about this 4 Mb of DNA (0.1% of the genome) on the short arm of chromosome 6 than about any other region of similar size. Among the 80 or so MHC genes found so far are several clusters with related functions in antigen processing and presentation. In addition to its importance in immunology, the MHC is a useful model for investigating gene organization, polymorphism, linkage disequilibrium and recombination. A large number of diseases, many of the autoimmune type, are associated with the region.

Animals↗
PubMed1993

Conservative evolution of the Mbc-DP region in anthropoid primates.

To determine the organization of the DP region in the Mbc of anthropoid primates, we constructed contig maps from cosmid clones of the chimpanzee and orangutan, representatives of the infraorder Catarrhini, as well as of the cotton-top tamarin, a representative of the infraorder Platyrrhini. We found the maps to be remarkably similar to each other and to the previously published map of the human DP region. In each of the four species, the DP region consists of four loci arranged in the same order (DPB2 . . . DPA2 . . . DPB1 . . . DPA1) and in the same transcriptional orientation (tail-to-tail). The regions in the four species are of approximately the same length and many of the restriction sites are shared between species. The inserts of most Alu elements, of a ribosomal protein pseudogene, and of an IgC epsilon-like pseudogene are found in corresponding positions in all four species. The data indicate that the human-type organization of the DP region was established before the divergence of the Catarrhini and Platyrrhini lines more than 37 million years ago and that it has remained principally intact since that time. This conservation of the DP region is in striking contrast to the evolutionary instability of certain other Mbc regions, in particular those occupied by the DRB or C4 and CYP21 loci. We interpret the stability of the DP region as an indication that the region is being phased out functionally.

Animals↗
PubMed1993

Mouse mammary tumor virus-mediated T-cell receptor negative selection in HLA-DRA transgenic mice.

Products of specific mouse Mtv genes expressed in association with mouse MHC class II products cause the deletion of T cells expressing particular TCR V beta gene segments. These endogenous deletion ligands have been termed superantigens due to their ability to negatively select entire T-cell families, as defined by V beta-chain usage. In most cases, deletion is preferentially effected through interaction of the Mtv ligand with H-2E products. Although human DR alpha shares only 75% identity with the E alpha chain of H-2E, it has previously been shown to substitute for the mouse homologue in its capacity to induce the deletion of V beta 11- and V beta 17a-bearing T cells. In the present study, we have undertaken a more comprehensive analysis of the interaction of mixed DR alpha/E beta pairs with various endogenous Mtv integrants in various mouse backgrounds, leading to negative selection of particular V beta families. We show in this paper that transgenic DR alpha/E beta can also efficiently interact with products of Mtv-7, causing deletion of both V beta 6+ and V beta 7+ cells. Deletion of V beta 11+ T cells in DRA transgenic mice carrying Mtv-8 and -9, however, was less efficient than in control H-2Ea transgenic mice. These data and those from other MHC transgenic mouse studies show that while the class II alpha chain can influence the interaction with superantigen, it is the identity of the beta chain that seems to be critical.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Frequent loss of heterozygosity on chromosome 6 in human ovarian carcinoma.

Investigation of genetic changes in tumours by loss of heterozygosity (LOH) is a powerful technique for identifying chromosomal regions that may contain tumour suppressor genes. LOH has been described on chromosome 6 in ovarian carcinoma using restriction fragment length polymorphism analysis with a small number of probes. We studied 29 ovarian carcinomas with 19 probes mapping to chromosome 6. Sixteen of the 29 tumours showed LOH on 6q (55%). Of these 16, 63% showed loss of all informative markers on that arm. One tumour showed loss of 6q24-qter, localising the putative tumour suppressor gene to that region. Loss on 6p was 28% overall. However, using three dinucleotide repeat primer pairs from 6p to study LOH in seven selected tumours, LOH was demonstrated at both 6p22.3-pter and at 6p12-6p22. These results confirm that 6q harbours a tumour suppressor gene of relevance to ovarian carcinoma and suggest that there may also be a similar gene(s) on 6p. By Southern analysis, there was no evidence of genomic rearrangements of the oestrogen receptor gene, located at 6q25.1. LOH on 6q was more common in high than low grade tumours. The relevance of our findings to previous work in ovarian cancer and other solid tumours is discussed.

Chromosome Deletion↗
PubMed1993

Loss of heterozygosity and amplification on chromosome 11q in human ovarian cancer.

The 11q13 chromosomal region encodes oncogenes relevant to a variety of human cancers as well as a tumour suppressor gene implicated in multiple endocrine neoplasia type 1. In addition, high affinity folate receptor (FOLR1), which maps to 11q13.3-13.5, is expressed at an elevated level on the surface of over 80% of nonmucinous epithelial ovarian cancers. Further telomeric, 11q breakpoints are found in many cancers. We studied the involvement of 11q markers in ovarian cancer by looking for tumour-specific loss of heterozygosity (LOH), as well as amplification or rearrangements that might explain the overexpression of FOLR1. Twenty eight epithelial ovarian cancers, along with lymphocyte DNA from the same individual were used for Southern blotting with polymorphic probes from 11q. PCR primers from 11q23.3 were also used. The 11q13 band was amplified in four out of 28 cancers. The amplicon included the probe D11S146 as well as FGF3 (formerly INT2) and FOLR1 in one out of these four cases, thus crossing the bcl1 translocation breakpoint. LOH was seen in three out of 16 cases with FGF3 (11q13). A much higher frequency of LOH (8/12) was found at 11q23.3-qter, implying the presence of a tumour suppressor gene in this region.

Chromosome Deletion↗
PubMed1993

Fine mapping of the human MHC class II region within chromosome band 6p21 and evaluation of probe ordering using interphase fluorescence in situ hybridization.

Eight previously well-characterized and mapped probes derived from the human major histocompatibility complex (MHC) class II region were used to investigate the advantages and limitations of fluorescence in situ hybridization (FISH) techniques for fine mapping. The class II region of the MHC was localized within subband 6p21.31 by in situ hybridization on metaphase chromosomes that were banded by immunofluorescence staining with an antibody against 5-bromodeoxyuridine (BrdU). Ordering of probes that were separated by up to 900 kb was achieved by simultaneous hybridization of two or three probes on interphase nuclei. Three-color FISH proved to be an excellent method for ordering of probes within distances of 200-1,000 kb. Under certain conditions, closer probes could be ordered by comparing measured distances between their hybridization signals in interphase nuclei. A linear correlation between measured interphase distances and kilobase distances was observed up to 500 kb. With increasing distances, the measurements become more inaccurate due to chromatin folding.

Chromosome Mapping↗
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↗