Molecular genetics of the HLA system.
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Amino Acid Sequence↗
Biomedical subjects
J Trowsdale
Publications and source records attributed to J Trowsdale.
Expression of alkaline phosphatase from cloned human tumor cell lines in human/mouse somatic cell hybrids.
Many human tumor cell lines produce alkaline phosphatases (APs) whose expression is normally not associated with the tissue where the tumor originated. The control of high level AP expression in three cloned human tumor cell lines was examined after fusion to murine fibroblast cell lines. The human tumor cell lines studied were: HeLa 71.5, producing term placental AP, D98.8, producing fetal intestinal AP and Hs578T.12, producing a liver-like AP. After fusion to the murine fibroblast cell lines IT22 or CL1D and propagation of the hybrids, the presence of human chromosome markers and the levels of AP were determined. Hybrids of either murine line fused to HeLa 71.5 or D98.8 cells never gave rise to proliferating clones with significant levels of their respective APs, even when several hundred hybrids were examined, in situ, at early and later stages of proliferation. Consequently, the abundant AP production normally found in HeLa 71.5 and D98.8 cells may require products from a number of chromosomes or the mouse cytoplasm is able to extinguish AP expression. Hybrids formed between the Hs587T cell line and either murine line continued to express liver-like AP. This expression was maintained in HAT selection medium and eliminated with bromodeoxyuridine counter selection. This result suggests that the control of liver-like AP in human tumors differs from that of the full-term placenta of fetal intestine.
Alkaline Phosphatase↗
A monoclonal antibody to intestinal alkaline phosphatase made against D98/AH-2 (HeLa) cells.
A monoclonal antibody (AAP1) to human intestinal alkaline phosphatase (ALP) was produced by immunizing a mouse with D98/AH-2 (HeLa) cells, which produce the enzyme ectopically. The antibody, which did not inhibit enzyme activity using p-nitrophenyl phosphate as the substrate, was of the IgG2A class and did not show complement-dependent cytotoxicity. In trace binding assays AAP1 bound only to cells that expressed an intestinal-like form of human ALP, including some human intraspecific (D98/AG-2 x human lymphocyte or fibroblast) hybrids. Immunoprecipitation of immune complexes from cell-free extracts of D98/AG-2 cells, using protein A containing S. aureus and AAP1 antibody, resulted in precipitation of all the ALP activity. The precipitated material had a subunit molecular weight of 80,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. In non-denaturing conditions, AAP1 antibody prevented the migration of ALP activity into the gel when cell-free extracts were made from human adult or fetal intestine, or D98/AH-2 cells. Similarly, AAP1 could be used to precipitate ALP activity from these extracts but not from extracts of human liver, kidney or placenta.
Alkaline Phosphatase↗
Expression of HLA-A, -B, and -C and beta 2-microglobulin antigens in human choriocarcinoma cell lines.
Immunoprecipitation of [35S]methionine-labeled cell extracts and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that cells from two human choriocarcinomas JaR and BeWo contained beta 2-microglobulin. The JaR cells, and one subline of BeWo, did not express surface HLA-A, -B, and -C (HLA-ABC) antigens, nor did JaR contain the antigens when cell-free extracts were measured in a sensitive radioimmunoassay. It may be concluded that the lack of expression of HLA-ABC antigens in choriocarcinoma (and in the trophoblast) is not a result of lack of beta 2-microglobulin, as is the case for the Burkitt's lymphoma cell line, Daudi.
Animals↗
Synthesis of HLA antigens from membrane-associated messenger RNA.
Proteins encoded by genes in the human major histocompatibility region, HLA, are present on the plasma membranes in the cells of most tissues. We have isolated messenger RNA (mRNA) from lymphoblastoid cells and studied its templating activity for HLA polypeptides by using an mRNA-dependent cell-free translation system and immunorecipitation with specific antisera. We estimate the sizes of the mRNA to be 1,700, 1,300, and 900 nucleotides for HLA-A, -B, and -C (HLA-ABC), HLA-DR, and beta 2-microglobulin, respectively. These sizes are consistent with our inability to detect larger polypeptide precursor for more than one HLA-ABC or HLA-DR antigen. Subcellular fractionation into cytosol and membrane fractions indicated that mRNA for the HLA-ABC and HLA-ABC and HLA-DR polypeptides was found predominantly in the membrane fraction. The beta 2-microglobulin mRNA appeared to be divided into roughly equal portions between both fractions. Nevertheless, we believe that in the cell, beta 2-microglobulin is synthesized in the rough endoplasmic reticulum, and that, because the protein is relatively small, the mRNA is released from the polyribosomes during the isolation procedure. We conclude that the properties of the HLA region and the beta 2-microglobulin mRNA are consistent with current models for the synthesis and membrane insertion of eukaryotic cell surface proteins. Our findings do not suggest the existence of multigenic precursor polypeptides for HLA antigens in contrast to those for several viral membrane glycoproteins.
Cell Line↗
Genetic analysis of a class of polymyxin resistant partial revertants of stage O sporulation mutants of Bacillus subtilis: map of the chromosome region near the origin of replication.
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Bacillus subtilis↗
New cluster of ribosomal genes in Bacillus subtilis with regulatory role in sporulation.
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Bacillus subtilis↗
Evidence that spo0A mutations are recessive in spo0A-/spo0A+ merodiploid strains of Bacillus subtilis.
The spo0A locus contains two types of closely linked mutations that block sporulation at stage 0: spo0A mutations (the most pleiotropic of the stage 0 markers) and spo0C mutations. It was previously thought that spo0A mutations were dominant in merodiploids of Bacillus subtilis, whereas spo0C mutations were recessive. We have shown that spo0A mutations were recessive when spo0A-/spo0A+ merodiploids were made in the genetic backgrounds of strains that were resistant to antibiotic produced by the wild-type strain. Reinvestigation of cultures of spo0A-/spo0A+ merodiploids constructed in the antibiotic-sensitive spo0A strain showed that they contained the spo0A allele at a low frequency, and they produced very few haploid Spo- segregants. These facts indicated that the cultures contained mostly homogenotic (spo0A+/spo0A+) cells. The reason for the poor survival of the spo0A-/spo0A+ merodiploids in the genetic background of the antibiotic-sensitive strain was not clear, but several possible explanations were given. It may have been related to the diploid state of other genes in the same merodiploid cells. The previous indication that spo0A mutations were dominant seems to have been based on the properties of a rare class of Spo- segregants that were probably selected in the presence of antibiotic.
Anti-Bacterial Agents↗
Differences in the genetic structure of Bacillus subitilis strains carrying the trpE26 mutation and strain 168.
It was previously shown that in strains of Bacillus subtilis bearing the trpE26 mutation a chromosome segment (from trpD to ilvA) is translocated to a position near the thr region. Further PBS1-mediated transduction data have now revealed that these strains also possess an inversion of part of the chromosome from the origin of replication, down to the tre locus on one side and the cysB locus on the other. These data concern evidence of linkage of tre-12- to markers in the translocation (hisH2, tyrA1, and metB3) as well as linkage of the cysB3 marker to thi-86, gly-133, and catA. They explain the previously observed absence of linkage of markers in the translocated segment to cysB3. The model proposed for the formation of merodiploids in trpE26 strains, which calls for the fusion of two genetic elements, is not incompatible with this new finding.
Bacillus subtilis↗
Evidence for the Translocation of a Chromosome Sement in Bacillus subtilis Strains Carrying the trpE26 Mutation.
The replication order of markers was studied in Bacillus subtilis strains bearing the trpE26 mutation by the use of the density transfer technique. An important difference in this order was observed in comparison with that of strain 168 T-. All markers tested of a chromosome segment extending from trpD to ilvA replicated early, after purB6 and before thr-5. Two markers flanking this region, trpE8 and citK7, replicated late as usual. The results suggested that this segment was shifted in trpE26 strains to a region closer to the origin of replication. PBS-1-mediated transduction crosses corroborated this hypothesis and revealed the position of the translocated segment. (i) Linkage was demonstrated for markers in the segment (hisH2, tryA1, met B3, ilvA2) to thr-5 and ald; (ii) aroB2 and citK7 were found to be linked; and (iii) linkage of cysB3 to thr-5 was lost in trpE26 strains. These findings made it possible to account for the characteristics of the trpE26 mutation and to propose a model explaining the fact that all trpE26+ transformants or transductants are merodiploid. The model calls for fusion of two genetic elements: two independent chromosomes, or two arms of a replicating structure. The resulting chromosome bears a long tandem duplication. Most of the features of this system of merodiploid formation can be interpreted by use of this model: the segregation pattern of the diploids, the stabilization of the unstable clones, and the length of the duplicated region. A relatively stable diploid strain was also studied by the density transfer technique. The data show that it remained diploid for the region corresponding to the translocated segment and are in agreement with the structure predicted by the model.
Bacillus subtilis↗
Isolation, characterization, and mapping of Bacillus subtilis 168 germination mutants.
After mutagenesis with nitrosoguanidine, germination mutants of Bacillus subtilis 168 were selected by killing, with heat, spores that germinated at 42 C and collecting survivors at 30 C. The germination properties of nine mutants variously affected in amino acid biosynthesis and sugar utilization were studied in detail. They were divided into two groups: (i) Ger-ALA mutants, failed to germinate in 10 mM L-alanine but germinated in complex media (some of these mutants were temperature sensitive); (ii) Ger-PAB mutants, germinated poorly, even in complex media, suggesting that they were blocked in important germination functions. All the mutants failed to germinate in L-alpha-amino-n-butyrate or L-valine (including temperature-sensitive mutants only at the restrictive temperature) showing that there is a step necessary for germination affected by all three acids. The mutants had normal growth rates, indicating that the defective gene products were specific for germination functions. These defects were not identified. Eight of the mutants were mapped by transduction with phage PBS-1. The recombinants were scored either by observations, by microscopy of phase darkening of the spores, or by a plate test involving the reduction of tetrazolium by heated colonies of spores. Five of the mutations, of at least three phenotypes, were between thr-5 and cysB3 away from all the sporulation markers that have been previously mapped. A linked ald (alanine dehydrogenase) locus was on the other side of thr-5. The other Ger markers were located in at least two additional positions. Auxotrophic strains that were used for mapping germinated normally, but germination of the Ger mutants differed slightly in different genetic backgrounds.
Bacillus subtilis↗
The mouse Dap10 gene.
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Amino Acid Sequence↗
Immunology. Molecular biology of the major histocompatibility complex.
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Animals↗
Functional expression of HLA-DP genes transfected into mouse fibroblasts.
The HLA class II antigens are a highly polymorphic family of dimeric cell-surface glycoproteins, expressed predominantly on the surface of immunocompetent cells. They are intimately involved with the induction of the T-cell response to extrinsic antigen and are important predisposing factors for a wide spectrum of autoimmune diseases. We describe here the expression of a class II product from the HLA-DP (new WHO nomenclature, formerly SB) subregion after transfer of cloned genes into mouse fibroblasts. The transfected DP antigen is recognized by several HLA class II monoclonal antibodies and, though present in a mouse cell background, is able to function in the presentation of influenza antigen to cloned DP-restricted human T lymphocytes.
Animals↗
Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters.
Class I molecules of the major histocompatibility complex (MHC) bind and present peptides derived from the degradation of intracellular, often cytoplasmic, proteins, whereas class II molecules usually present proteins from the extracellular environment. It is not known how peptides derived from cytoplasmic proteins cross a membrane before presentation at the cell surface. But certain mutations in the MHC can prevent presentation of antigens with class I molecules. In addition, mutations possibly in the MHC can affect presentation by class II molecules. Here we report the finding of a new gene in the MHC that might have a role in antigen presentation and which is related to the ABC (ATP-binding cassette) superfamily of transporters. This superfamily includes the human multidrug-resistance protein, and a series of transporters from bacteria and eukaryotic cells capable of transporting a range of substrates, including peptides.
Amino Acid Sequence↗
Complexity in the major histocompatibility complex.
The human major histocompatibility complex (MHC) is one of the most intensively studied regions of the human genome, containing over 70 known genes and spanning about 4 million base pairs (4 Mbp) of DNA on chromosome 6p21.3 (Klein, 1986). It can be divided up into three regions: the class I region (telomeric), the class II region (centromeric), and the class III region (between class I and II), which includes the complement component genes C2, C4, and Bf (Trowsdale & Campbell, 1988). The MHC has been mapped in detail using pulse field gel electrophoresis (PFGE) and by cloning in yeast artificial chromosome (YAC) and cosmid vectors, revealing long stretches of DNA between the regions as well as between individual class I and class II genes. Novel genes, that have no sequence relationships with class I, class II or complement components, have recently been found in these areas, and we will present an update on these after reviewing the more established loci.
Chromosome Mapping↗