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D D Chaplin

Publications and source records attributed to D D Chaplin.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning of the murine IL-1 beta converting enzyme cDNA.

IL-1 beta is a potent modulator of immune and inflammatory responses. Murine IL-1 beta is initially synthesized as an inactive 33-kDa pro-molecule that is activated by proteolytic cleavage between Asp-117 and Val-118 to generate the 17-kDa mature IL-1 beta protein. This cleavage is catalyzed by a specific protease that has been designated the IL-1 beta converting enzyme (or IL-1 beta convertase). We have used a human IL-1 beta convertase cDNA to isolate murine convertase cDNA from a WEHI-3 library. These cDNA predicted that the murine convertase is a 402-residue protein. Overall, the murine convertase showed 71% nucleotide and 62% predicted amino acid sequence identity with the human convertase. Southern blot analysis of interspecific backcross mice indicated that the murine IL-1 beta convertase is encoded by a single copy gene located on murine chromosome 9. The murine convertase showed broad constitutive expression, being detected in mononuclear phagocyte and T lymphocyte cell lines as well as in spleen, heart, brain, and adrenal glands. The expression of the murine convertase in mononuclear phagocytes was up-regulated by treatment with LPS or rIFN-gamma. These studies establish that the IL-1 beta convertase is an evolutionarily conserved, widely expressed enzyme that can be regulated at a pretranslational level.

Amino Acid Sequence↗
PubMed1992

Cloning and physical mapping of the HLA class I region spanning the HLA-E-to-HLA-F interval by using yeast artificial chromosomes.

The HLA class I genes are located within a 2-million-base pair (2-Mbp) region constituting the telomeric half of the human major histocompatibility complex. The large majority of the class I sequences, including the HLA-A, -E, -F, and -G genes, is found within the telomeric 1 Mbp. We report here the isolation and characterization of yeast artificial chromosome (YAC) clones that span a contiguous region of greater than 1.2 Mbp and include 14 of the 18 characterized class I sequences. Restriction enzyme mapping and the use of locus-specific probes have allowed all of the class I genes and sequences to be ordered and positioned within the region. In addition, the transcriptional orientation of the four class I genes has been determined. Using probes derived from the ends of YAC inserts and from class I pseudogenes, we describe a highly polymorphic region between the HLA-A and HLA-G genes. This region appears to be deleted in certain HLA haplotypes, shortening the distance between HLA-A and HLA-G by greater than 50 kilobase pairs (kbp). As part of the characterization of the YAC clones, unique sequence probes derived from the ends of each YAC insert were identified. When combined with probes derived from HLA genes and pseudogenes, 25 locus-specific probes spanning the 1.2-Mbp region have been identified for an average of 1 probe every 48 kbp.

Chromosomes, Fungal↗
PubMed1991

cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments.

Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.

Base Sequence↗
PubMed1991

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha.

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.

Amino Acid Sequence↗
PubMed1991

Interleukin 1 is processed and released during apoptosis.

Interleukin (IL-) 1 alpha and 1 beta are synthesized as 31- to 34-kDa pro molecules. They are released from monocytes and macrophages as proteolytically processed 17-kDa mature molecules that bind with high affinity to specific receptors on target cells. IL-1 is not released via the classic secretory pathway. The pro molecules are synthesized as cytosolic proteins without signal peptides. Although the proteases that convert the pro molecules to the mature forms are cytosolic enzymes, processed IL-1 is not detected associated with the cell but is found only in culture supernatants. We demonstrate here that release of IL-1 is efficiently induced by cell injury. When the injury causes cellular necrosis, IL-1 alpha is released as a mixture of unprocessed and processed molecules but IL-1 beta is released exclusively as the biologically inactive pro form. In contrast, when cells undergo apoptosis, maturation of both IL-1 alpha and IL-1 beta is efficient. When apoptosis is rapid, as in macrophages that are targets for allospecific cytotoxic T lymphocytes, processing is observed to occur intracellularly. These findings suggest that cell injury is an important physiologic stimulus for release of IL-1. The nature of the injury profoundly affects the forms of IL-1 that are released.

Animals↗
PubMed1991

Release of IL-1 from mononuclear phagocytes.

IL-1 alpha and -beta are 31- and 34-kDa cytokines produced by stimulated monocytes, macrophages, and a variety of other cells. These proteins are thought to function primarily as intercellular mediators and can be detected in plasma and the supernatants of cultured cells; however, IL-1 alpha and -beta contain no identifiable signal peptides and are not secreted via the classical secretory pathway. To understand the mechanism of IL-1 release, we have analyzed IL-1 production by LPS-stimulated mononuclear cells. IL-1 was quantified by bioassay, immunoprecipitation, and ELISA. Of these techniques, only immunoprecipitation permitted the quantitative detection of intracellular pro-IL-1. Both the full-length pro-forms and proteolytically processed mature forms of IL-1 were detected in culture supernatants; however, for macrophages the released material represented less than 5% of the total IL-1 alpha and -beta synthesized. Freshly isolated human monocytes released a higher fraction of their total IL-1 (up to 22%): however, monocytes cultured in vitro for 24 h showed very little fractional release, similar to macrophages. Nonspecific release of intracellular contents was determined by measurement of release of lactate dehydrogenase activity and was found to parallel IL-1 release. In fact the higher release of IL-1 from freshly cultured human monocytes correlated also with an increase in the release of lactate dehydrogenase. We conclude that, in cultured LPS-stimulated monocytic cells, IL-1 is not released via a novel secretory pathway, but exits the cell via a nonspecific pathway, most likely as a consequence of cellular injury.

Animals↗
PubMed1991

Isolation and characterization of yeast artificial chromosome clones linking the HLA-B and HLA-C loci.

A 290-kilobase-pair chromosomal segment containing the genes encoding the human class I major histocompatibility complex molecules HLA-B and HLA-C as well as a class I pseudogene has been isolated on three overlapping yeast artificial chromosome (YAC) clones. One YAC clone contains both the HLA-B and HLA-C genes. These loci are located approximately 85 kilobase pairs apart, each in close association with a CpG island. Southern blotting and nucleotide sequencing showed no evidence of alteration of the structure of the cloned DNA in the YACs. End fragments from the YAC inserts have been isolated and used to confirm the overlaps between clones. These fragments can also serve as polymorphic markers for structural analysis of the major histocompatibility complex. Our data show that YAC cloning offers an attractive alternative for analysis of the structures of large gene complexes such as HLA.

Amino Acid Sequence↗
PubMed1991

Generation of monoclonal antibodies to murine IL-1 beta and demonstration of IL-1 in vivo.

The role of murine IL-1 beta in vitro and in vivo has not been defined. We describe here the production of neutralizing and immunoprecipitating mAb and polyclonal antibodies specific for murine IL-1 beta and their application to a characterization of the murine IL-1 beta protein. Immunization of either hamsters or rabbits with the recombinant mature form of murine IL-1 beta emulsified in CFA elicited antisera and hamster mAb that only recognized denatured IL-1 beta. In contrast, immunization with rIL-1 beta adsorbed to alum resulted in the generation of neutralizing and immunoprecipitating rabbit and hamster antisera and hamster mAb. All of the mAb recognize both the pro-form of IL-1 beta and the mature bioactive form produced by cultures of murine peritoneal macrophages. Using these antibodies, we demonstrate that approximately half of the IL-1 activity present in supernatants of LPS-treated cultured mouse macrophages is composed of IL-1 beta. Additionally, IL-1 beta as well as IL-1 alpha can be detected in the plasma of LPS-treated mice. These studies, therefore, demonstrate the production of IL-1 beta both in vitro and in vivo.

Animals↗
PubMed1991

Molecular linkage of the HLA-DR, HLA-DQ, and HLA-DO genes in yeast artificial chromosomes.

Eight major histocompatibility complex (MHC) class II loci and the newly defined Y3/Ring 4 locus were isolated in overlapping yeast artificial chromosome (YAC) clones defining a 420-kb segment of human chromosome 6p21.3. YAC B1D12 spanning 320 kb contained seven of these loci from HLA-DRA to HLA-DQB2. A 330-kb YAC, A148A7, spanned from the HLA-DQA1 locus through the Y3/Ring 4 locus and extended at least 130 kb centromeric of YAC B1D12. Southern blotting demonstrated that YAC B1D12 derived from the HLA-DR3 haplotype and that YAC A148A7 derived from the HLA-DR7 haplotype of the heterozygous library donor. A third 150-kb YAC, A95C5, lay within this contig and contained only the HLA-DRA locus. A fourth 300-kb YAC, A76F11, was isolated by chromosome walking from the telomeric end of YAC B1D12. Probes isolated from the ends of the YAC genomic inserts have been used to confirm overlaps between the clones. These analyses demonstrated that the centromeric end of YAC A76F11 used the same genomic EcoRI cloning site as the telomeric end of YAC A95C5. YAC B1D12 used an EcoRI site only 2.1 kb telomeric of the aforementioned EcoRI site. These data suggest that certain EcoRI sites are used preferentially during construction of the library. These YACs complete the linkage of the DR and DQ subregions of the HLA complex in cloned DNA and provide the substrate for precise analysis of this portion of the class II region.

Base Sequence↗
PubMed1991

Different isozymes of mouse 11 beta-hydroxylase produce mineralocorticoids and glucocorticoids.

We have isolated and characterized two isozymes of mouse steroid 11 beta-hydroxylase (11 beta-OHase), designated 11 beta-OHase and aldosterone synthase (AS). Physical mapping of overlapping cosmid and phage isolates defined two genes (designated Cyp11b-1 and Cyp11b-2 in the standard nomenclature for cytochrome P450 genes) that are oriented in the same direction and separated by approximately 8 kilobase pairs of DNA. The two genes are highly homologous in their coding regions, with 84% nucleotide identity and 86% predicted amino acid identity. In regions where the sequences of the rat 11 beta-OHase and AS genes diverged most widely, the mouse sequences also differed significantly, thereby identifying putative mouse 11 beta-OHase and AS genes. Both genes were mapped to chromosome 15 by analyzing restriction fragment length variations in a panel of DNA samples from an interspecific cross. To determine the functional properties of the 11 beta-OHase and AS proteins, we transfected COS-7 cells with plasmids that expressed the proteins encoded by the 11 beta-OHase and AS genes. When expressed in transfected COS-7 cells, the 11 beta-OHase protein converted deoxycorticosterone to corticosterone but did not produce aldosterone. Consistent with its postulated role in mineralocorticoid biosynthesis, the product of the AS gene efficiently synthesized aldosterone. We next studied the expression of these two isozymes in Y1 adrenocortical tumor cells and in the intact mouse adrenal gland. Although Y1 cells otherwise resemble zona fasciculata cells and express the 11 beta-OHase gene at high levels, transcripts encoded by the AS gene were detected at levels approximately 10-fold lower than the 11 beta-OHase transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Neoplasms↗
PubMed1990

Genetic variability at the human tumor necrosis factor loci.

Variability in the structure of the human tumor necrosis factor (TNF-alpha) or lymphotoxin (TNF-beta) genes may contribute to the functional polymorphism of the HLA gene complex. We have characterized an allelic restriction fragment length polymorphism (RFLP) of the TNF-beta gene by using the restriction endonuclease NcoI. Digestion of genomic DNA with NcoI and Southern blotting by using TNF-alpha gene probes show 5.4-kb and 10.5-kb hybridizing fragments. In Caucasian populations, the 10.5-kb fragment is present in 64 to 72% of haplotypes. The polymorphic NcoI site is located within the first intron of the TNF-beta gene. Additional restriction fragment variability was demonstrated by digestion with AccI; however, this restriction fragment variability was not allelic in nature. Rather, it was a consequence of variable DNA methylation at AccI sites within and upstream of the TNF-beta gene. In peripheral blood leukocytes, methylation of the TNF-beta AccI sites was greatest in neutrophils (TNF-beta nonproducers), and lowest in T lymphocytes (the major producers of TNF-beta). These results suggest strongly that variation in DNA methylation may play an important role in regulation of the expression of the TNF-beta gene.

Azacitidine↗
PubMed1990

Compartmentalization of mammalian proteins produced in Escherichia coli.

We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.

Amino Acid Sequence↗
PubMed1990

Identification and sequence analysis of four complement factor H-related transcripts in mouse liver.

Mouse liver RNA analyzed by Northern blotting with a full-length complement factor H cDNA probe demonstrates the 4.4-kilobase (kb) H mRNA as well as three additional hybridizing species of 3.5, 2.8, and 1.8 kb, respectively. Further characterization of these alternative transcripts was pursued by isolation of additional cDNAs from a liver library using a full-length H probe. Twelve clones homologous to but distinct from H were isolated, analyzed by restriction mapping, and divided into four classes, A, B, C, and D, based on their sequences. Clones from classes A, B, and C all contained nearly identical 5'-untranslated regions and leader sequences that differed from H at more than 50% of their nucleotide positions. The 5'-untranslated and leader sequences of the class D clone were unrelated to the corresponding regions of H or the class A, B, or C clones. The remaining portions of the H-related cDNAs were made up of short consensus repeats, 7 in class A, 4 in class B, 13 in class C, and 5 in class D. To determine the relationship between the H-related transcripts and the cDNA clones, Northern blots of liver RNA were analyzed by hybridization with two probes, one specific for the class D cDNA and the other reacting specifically with the class A, B, and C cDNAs. The class A/B/C probe detected transcripts of 3.5, 2.8, and 1.8 kb in liver RNA, and the class D probe hybridized to a distinct 1.8-kb message. Additionally, a cosmid genomic library was screened with H cDNA, and nine H-related clones were isolated. They spanned a region of approximately 120 kb, defining at least two discrete H-related gene loci. These results identify new members of the super-family of C3b/C4b binding protein genes.

Amino Acid Sequence↗
PubMed1990

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene.

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Factor H.

While the mouse and human H proteins are structurally and functionally similar, they differ in their genetics. Whereas there is no evidence in humans for more than one gene; in mice the H locus is complex. Based on cDNA sequence and hybridization analysis of genomic cosmid clones, there are at least three distinct genes, all highly related to one another. The consensus repeating unit that comprises this molecule has obviously been duplicated numerous times, since it is present in many other molecules. Thus, it is not surprising to discover that there are several genes related to H in the mouse. A similar case has been described for two other members of this family. In humans, CR1 cDNA hybridizes to two distinct genomic clusters in the CR1 locus (Wong et al. 1989), and in mice, mCRY hybridizes to two regions in the genome, one on chromosome 1 and another on chromosome 8 (Aegerter-Shaw et al. 1987). It will be of interest to see if any other members of this family display as complex a genetic locus as murine H.

Animals↗
PubMed1990

Genomic organization of mouse Fc gamma receptor genes.

We have isolated and characterized the gene coding for the mouse Fc receptor that is termed Fc gamma RIIa. The gene contains five exons and spans approximately 9 kilobases. Unlike most members of the immunoglobulin gene superfamily, this gene utilizes multiple exons to encode its leader peptide. The first exon encodes the hydrophobic region of the signal sequence; the second exon, which contains only 21 base pairs, encodes a segment of the signal peptidase recognition site; and the beginning of the third exon encodes the predicted site of peptidase cleavage. The third and fourth exons each code for immunoglobulin-like extracellular domains. The fifth exon encodes the hydrophobic transmembrane domain and the cytoplasmic tail. Partial characterization of the Fc gamma RIIb gene indicates that it also contains multiple leader exons, including a 21-base-pair exon and two exons coding for homologous immunoglobulin-like extracellular domains. However, the Fc gamma RIIb gene uses four exons to encode its intracytoplasmic region. Analysis using contour-clamped homogeneous electric field (CHEF) gels indicates that the Fc gamma RIIa and Fc gamma RIIb genes are linked within 160 kilobases on mouse chromosome 1.

Amino Acid Sequence↗
PubMed1989

Molecular analysis of the murine C4b-binding protein gene. Chromosome assignment and partial gene organization.

Murine C4b-binding protein (C4BP) is a regulatory molecule in the classical pathway of complement. C4BP is composed predominantly of short consensus repeats (SCRs) approximately 60 amino acids in length, which contain a framework of conserved residues. The SCRs are found in many complement molecules and a growing number of noncomplement molecules as well and are a major structural feature of some of these molecules. To characterize the structure of the murine C4BP gene, a cosmid library constructed from Balb/c liver DNA was screened. Several nearly identical, overlapping clones were identified; however, none of the clones, alone or in combination, covered the entire C4BP gene. One clone (D26) was chosen for detailed analysis and found to contain all but the leader region, the first SCR, and the first half of the second SCR. The SCRs three through six were each encoded by single exons. Only the latter half of the second SCR was present on the clone, and it was encoded by a single exon, demonstrating that murine C4BP has a split SCR at the genomic level. Structural mapping of this portion of the gene demonstrates that the region extending from the second half of the second SCR through the nonrepeat and untranslated region spans approximately 12 kb; however, genomic Southern blot analysis suggests that the gene is between 20 and 30 kb in length. Analysis of the 3' genomic sequence demonstrates that this region of the gene has homology with SV-40 late (class II) RNA sequences. These sequences may play a role in 3' cleavage of the precursor RNA prior to polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗