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Biomedical subjects

J Trowsdale

Publications and source records attributed to J Trowsdale.

At least 217 records · Page 12Linked to original sources

MHC class II sequences of an HLA-DR2 narcoleptic.

Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 beta chains, along with DQw1 alpha and beta chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3' untranslated region of one of the DR2 beta genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 alpha or beta from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQ alpha. Partial protein sequences of both DQ alpha and beta from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR beta or DQ alpha/beta sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.

Alleles↗

Epitope recognition by a DP alpha chain-specific monoclonal antibody (DP11.1) is influenced by the interaction between the DP alpha chain and its polymorphic DP beta chain partner.

The HLA-DP alpha chain-specific monoclonal antibody DP11.1 binds only to the surfaces of cells types as DPw2 or DPw4 by primed lymphocyte typing. To investigate the molecular basis for this antibody binding specificity, we isolated a DPw3 alpha chain cDNA clone and compared its sequence to those of other published DP alpha chain alleles. Interestingly, the extracellular region of the DPw3 alpha chain was identical to the analogous regions of DPw2 and DPw4 alpha chains. Immunoblotting analysis confirmed that the DP11.1 epitope is conserved on denatured DP alpha chains associated with cells typed as DPw2, DPw3, and DPw4. Therefore the binding of antibody DP11.1 to its alpha chain epitope is influenced by the associations between the DP alpha chain and its polymorphic DP beta chain partner.

Amino Acid Sequence↗

Molecular genetics of the MHC.

Although some gaps remain, the major histocompatibility complex (MHC) has now been mapped in considerable detail in both humans and mice. From the results of DNA cloning and sequencing, there is now a detailed knowledge of which genes are expressed and the composition of the proteins encoded by them, as well as the structural basis of the polymorphism. The problem that can now be addressed is how the sequences of the polymorphic domains relate to the function of HLA molecules and to the diseases associated with the MHC.

Animals↗

Molecular variation of human major histocompatibility complex DQw3 beta-chains.

Histocompatibility leukocyte antigen DQ molecules exhibit polymorphism of both DQ alpha- and beta-chains. Histocompatibility leukocyte antigen-DQw3 is associated with both DR4 and DR5 and can be further subdivided by reactivity with the monoclonal antibody TA10. To determine the molecular nature of the DQ polymorphic alleles associated with the DR4 haplotype, we have sequenced and analyzed DQ alpha and beta cDNA clones obtained from a DR4, Dw4, DQw3 cell line which is TA10-positive. The DQ alpha-chain sequence was identical to previously published sequences from the DR4 haplotype, but the DQ beta sequence differed from published DR4-DQ beta sequences obtained from DQw3-positive TA10-negative cell lines by eight amino acids, six of which were located in the beta 1 domain. Thus, the TA10 serologic determinants reside on the DQ beta-chain. A TA10-specific oligonucleotide probe was constructed based on the DQ beta sequence, and its specificity was confirmed in a panel of TA10-positive and TA10-negative cell lines. An additional band was observed in Southern blotting experiments which may indicate a donor sequence for gene conversion.

Alleles↗

Linkage of TNF genes to the HLA-B locus.

Pulsed field gel electrophoresis was used to determine the location of the tumour necrosis factor (TNF) alpha and beta genes. They were shown to be linked to the HLA-B locus; analogous to their location in mouse, between the complement (class III) region and H-2D. However, the distance between the TNF genes and the class I region was much greater in man, namely about 260 kb, compared to 70 kb in the mouse. This finding may have implications for some HLA associated diseases.

Genes↗

The human homologue of the mouse t-complex gene, TCP1, is located on chromosome 6 but is not near the HLA region.

Southern blot analysis indicates that there are four sequences in the human genome related to the mouse t-complex gene Tcp-1. All four genes were cloned and partial sequencing showed that one of them was a functional gene, and the other three were pseudogenes. The human sequences were all approximately 90% related to each other and 82-89% related to the mouse Tcp-1a sequence. Human TCP1 cDNA clones from both fibrosarcoma and B cell lines confirmed that there was a single expressed gene. mRNA transcripts of different sizes were accounted for by two different polyadenylation signals. The human TCP1 gene shared some amino acid substitutions with the mouse t-complex allele (Tcp-1a) which were not found in Tcp-1b. The functional human TCP1 gene was mapped, using a panel of somatic cell hybrids, as well as in situ analysis, to the long arm of chromosome 6 at 6q23-qter and thus is not closely linked to the HLA complex on the short arm. For this reason and others it is unlikely that there is a human equivalent of the mouse t-complex.

Amino Acid Sequence↗

Identification of HLA-DP polymorphism with DP alpha and DP beta probes and monoclonal antibodies: correlation with primed lymphocyte typing.

Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP beta probes and a DP alpha probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of DPw1, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defined and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies.

Alleles↗

Sequence and evolution of HLA-DR7- and -DRw53-associated beta-chain genes.

cDNA clones representing products of the DR7 and DRw53 beta-chain genes were isolated from the human B-lymphoblastoid cell line MANN (DR7,DRw53,DQw2, DPw2). The DRw53 beta sequence was identical to a DRw53 beta sequence derived from cells with a DR4 haplotype. In contrast, the DR7 beta sequence was as unrelated to DR4 beta sequence as it was to other DR beta-related genes, except at the 3'-untranslated region. These results suggest that the DR7 and DR4 haplotypes may have been derived relatively recently from a common ancestral haplotype and that the DR4 and DR7 beta-chain genes have undergone more rapid diversification in their beta 1 domains, most probably as a result of natural selection, than have the DRw53 beta-chain genes. Short tracts of sequence within the DR7 and DRw53 beta 1 domains were shared with other DR beta sequences, indicating that exchanges of genetic information between beta 1 domains of DR beta-related genes have played a part in their evolution. Serological analysis of mouse L-cell transfectants expressing surface HLA-DR7 molecules, confirmed by antibody binding and allelic sequence comparisons, identified amino acid residues that may be critical to the binding of a monomorphic DR- and DP-specific monoclonal antibody.

Amino Acid Sequence↗

Molecular mapping of the human major histocompatibility complex by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis and "cosmid walking" have been used to establish a molecular map of the human major histocompatibility complex (MHC). We have isolated approximately equal to 230 kilobases (kb) of genomic DNA in overlapping cosmid clones covering the genes for the second and fourth components of complement (C2 and C4, respectively), factor B, and steroid 21-hydroxylase, and approximately equal to 82 kb of genomic DNA surrounding the genes for the tumor necrosis factors alpha and beta. Single-copy hybridization probes isolated from these cosmid clusters and probes for the known MHC gene loci were hybridized to Southern blots of genomic DNA that had been digested with infrequently cutting restriction endonucleases and separated on pulsed-field gels. The data obtained allowed the construction of a long-range genomic restriction map and indicated that the MHC spans 3800 kb. This map orients the MHC class III gene cluster with respect to the DR subregion; the C2 gene is on the telomeric side of the 21-hydroxylase B gene. In addition we have defined the positions of the genes for the tumor necrosis factors alpha and beta in the human MHC. Genes for the alpha chain of DR and 21-hydroxylase B are separated by at least 300 kb, while the distance between the genes for C2 and tumor necrosis factor alpha is 390 kb. The HLA-B locus lies approximately equal to 250 kb on the telomeric side of the tumor necrosis factor genes.

Chromosome Mapping↗

The human placental alkaline phosphatase gene and related sequences map to chromosome 2 band q37.

A human placental alkaline phosphatase (PLAP) cDNA was isolated from a lambda gt 10 library of the cell line HEp-2. Southern blots probed with a fragment of the cDNA clone showed that the human genome may contain more than one PLAP-related sequence. The PLAP probe showed person-to-person variation in banding pattern with a number of enzymes. Using a panel of human/rodent somatic cell hybrids the PLAP sequences were mapped to chromosome 2. In situ hybridization confirmed this assignment and localized the gene(s) to chromosome 2 band q37.

Alkaline Phosphatase↗

The molecular genetics of human chromosome 6.

Chromosome 6 contains several clinically important markers as well as classical enzyme loci, proto-oncogenes, and a growing number of anonymous DNA restriction fragment length polymorphisms (RFLPs). It is also of unique interest because of the location of the major histocompatibility complex (MHC) on the short arm, at 6p21.3. The MHC is one of the most detailed areas of the human genetic map to date and many important diseases, some of a suspected autoimmune aetiology, are associated with it.

Chromosome Mapping↗

Mapping of HLA genes using pulsed-field gradient electrophoresis.

The technique of pulsed-field gradient electrophoresis (PFGE) allows the determination of gene linkage relationships since DNA fragments up to 2 Mb can be separated. PFGE was employed to study linkage of class I, II and III genes belonging to the human major histocompatibility (HLA) complex. The results establish that the class II DO beta and DZ alpha genes are linked with the DP subregion, centromeric to the DQ/DX-DR-C4 chromosomal segment, and allow us to estimate the minimal length of the entire HLA complex.

Chromosome Mapping↗

Gene clusters and the evolution of the major histocompatibility system.

Gene clusters containing one or more sets of duplicated genes with related functions are probably the basic genetic functional units. The major histocompatibility systems, such as HLA and H2, are among the most complex gene clusters so far known and studied, and illustrate many of the features of their structure and evolution. They cover about one thousandth of the mammalian genome and include two major sets of cell surface products with different but related functions in the control of immune interactions, as well as genes for complement components and 21-hydroxylase. Molecular analysis has revealed an extraordinary complexity at the genetic level, reflecting a very long and involved evolutionary history. A description of the organization of the HLA system, especially the HLA-D region, and its function and polymorphism forms the basis for considering the evolution of such complex gene clusters.

Amino Acid Sequence↗

Regional assignments of three polymorphic DNA segments on human chromosome 15.

Hybridization of probe pDP151 (locus D15S2) to genomic human DNAs digested with EcoRI revealed allelic restriction fragments 9 and 11 kilobase-pairs (kb) in length. Hybridization of pDP151 to EcoRI-digested DNAs from 21 Chinese hamster X human hybrid cell clones containing different subsets of human chromosomes demonstrated cosegregation of the 9 and 11 kb EcoRI fragments with human chromosome 15. D15S2 and two other polymorphic loci previously mapped to chromosome 15--D15S1 and D15S6--were localized to specific regions on human chromosome 15. Eight Chinese hamster X human somatic cell hybrid clones derived from a human donor heterozygous for a balanced translocation between chromosomes 15 and 22 [t(15;22)(q14;q13.3); Oliver et al, Cytogenet Cell Genet 22:503-510, 1978] were studied. After digestion of human and hybrid DNAs with HindIII and Southern blotting, pDP151 (D15S2) and pMS1-14 (D15S1) hybridized to fragments of 4 and 4.5 kb, respectively. Further, pMS1-14 (D15S1) and p9-1a (D15S6) hybridized to EcoRI fragments of 3.5 and 3.2 kb. All fragments cosegregated with the der(22) derivative chromosome containing region 15q14----15qter. In situ hybridization of these probes to normal human chromosomes mapped the corresponding loci with greater precision: D15S1 to 15q15----15q21, D15S2 to 15q15----15q22, and D15S6 to 15q22----15q24.

Chromosome Mapping↗