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D D Chaplin

Publications and source records attributed to D D Chaplin.

At least 55 records · Page 3Linked to original sources
PubMed1995

A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene.

Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.

Animals↗
PubMed1995

Absence of lymph nodes in lymphotoxin-alpha(LT alpha)-deficient mice is due to abnormal organ development, not defective lymphocyte migration.

Mice homozygous for a targeted null mutation of lymphotoxin-alpha (LT alpha) are born without lymph nodes (LN) or Peyer's patches (PP) and with altered splenic architecture. To investigate the mechanism of failed LN organogenesis, we transferred bone marrow (BM) from Thy 1.2 LT alpha-deficient or Thy 1.2 wild type mice to lethally irradiated 8-12-week-old Thy 1.1 wild type recipients. Six to 10 weeks later, reconstitution of LN and spleen with Thy 1.2 cells was similar whether the BM was derived from LT alpha-deficient or wild type donors. In contrast, reconstitution of irradiated LT alpha-deficient mice with wild type BM did not induce the development of detectable LN, although reconstitution of the spleen occurred appropriately. The expression and regulation of the lymphocyte adhesion molecule L-selectin from the LT alpha-deficient mice appeared normal. These data indicate that LT alpha-dependent interactions must occur during development in order for LN genesis to take place; however, lymphocyte expression of LT alpha is not required for these cells to home to existing LN structures.

Animals↗
PubMed1994

Upstream NFIL-6-like site located within a DNase I hypersensitivity region mediates LPS-induced transcription of the murine interleukin-1 beta gene.

To define the cis-acting elements that regulate LPS-stimulated IL-1 beta gene transcription, we analyzed the murine IL-1 beta gene by digestion with DNase I. At least two hypersensitive sites were located between 2200 and 2600 bp upstream of the transcription start site in mononuclear phagocytes, but not in an IL-1 nonproducing immature T cell line. Specific DNA sequences required for LPS induction of IL-1 beta gene expression were identified within the DNase I hypersensitive (DH) region using transfection of reporter constructs that contained portions of the IL-1 beta 5'-flanking region. Two specific DNA sequences were targets for nuclear factor binding as assessed with use of electrophoretic mobility shift analysis (EMSA). One site contained a consensus sequence for NFIL-6 binding. Base substitutions within this NFIL-6 site resulted in virtual elimination of LPS-induced IL-1 beta gene transcription. Introduction of multimers of the NFIL-6-like sequence immediately 5' to homologous or heterologous promoters conferred LPS-induced transcription, indicating that this NFIL-6-like consensus site was a transcriptional activator. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) Abs identified both of these proteins in complexes formed between the NFIL-6-like element and mononuclear cell nuclear extracts. C/EBP delta (NFIL-6 beta) was not detected in complexes utilizing extracts from the IL-1 nonproducing T cell line. These data are consistent with the requirement for C/EBP beta (NFIL-6) and C/EBP delta (NFIL-6 beta) in the activation of murine IL-1 beta gene expression by endotoxin.

Animals↗
PubMed1994

P1 and cosmid clones define the organization of 280 kb of the mouse H-2 complex containing the Cps-1 and Hsp70 loci.

A 280 kilobase (kb) contig was isolated from mouse genomic P1 and cosmid libraries, using as probes human cDNA and genomic DNA fragments that map in the interval between the second component of complement and tumor necrosis factor genes of the HLA complex. The clone contig demonstrates synteny of eleven mouse genes that are homologous to genes initially mapped within the human major histocompatibility complex. These include the mouse homologs of BAT2 (HLA-B-associated transcript 2) through BAT9 and also three HSP70-related genes. Five P1 clones form a contig of 240 kb that spans from BAT9 through BAT3. Twelve cosmid clones are arranged in three contigs that confirm most of the structure of the P1 contig and link the mouse BAT3 homolog to the BAT2 homolog approximately 15 kb farther telomeric. Polymorphic DNA markers within the cloned region were used to map the cleft palate susceptibility-1 (Cps-1) locus to the interval between Hsp70.1 and BAT6 (valyl-tRNA synthetase). This refines the location of the Cps-1 locus to a 45 kb region contained in the H2-124 P1 insert.

Adenosine Triphosphatases↗
PubMed1994

An NFIL-6 sequence near the transcriptional initiation site is necessary for the lipopolysaccharide induction of murine interleukin-1 beta.

We have analyzed the interleukin-1 beta (IL-1 beta) promoter region near the cap site. Specific DNA sequences required for lipopolysaccharide (LPS) induction within this region were identified using transfection of reporter plasmids that contained portions of the proximal IL-1 beta 5'-flanking sequence. An LPS-responsive activation area was localized between nucleotides -50 to -100, and down-regulating sequences were present between nucleotides -100 and -2,111. A NFIL-6 site between -92 and -84 was identified in the functionally active region. Base substitutions within this single NFIL-6 site in the context of a 4.1-kb IL-1 beta promoter segment resulted in dramatic reduction of LPS-induced gene transcription. Introduction of multimers of this NFIL-6 sequence immediately 5' to minimal homologous or heterologous promoters conferred LPS inducibility in each case. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) antibodies identified both of these proteins in complexes formed between the NFIL-6 site and mononuclear cell nuclear extracts. These data show that the proximal NFIL-6 site is required for the activation of murine IL-1 beta gene expression by endotoxin.

Animals↗
PubMed1993

Genes in one megabase of the HLA class I region.

To define the gene content of the HLA class I region, cDNA selection was applied to three overlapping yeast artificial chromosomes (YACs) that spanned 1 megabase (Mb) of this region of the human major histocompatibility complex. These YACs extended from the region centromeric to HLA-E to the region telomeric to HLA-F. In addition to the recognized class I genes and pseudogenes and the anonymous non-class-I genes described recently by us and others, 20 additional anonymous cDNA clones were identified from this 1-Mb region. We also identified a long repetitive DNA element in the region between HLA-B and HLA-E. Homologues of this element were located at several sites in the human genome outside of the HLA complex. The portion of the HLA class I region represented by these YACs shows an average gene density as high as the class II and class III regions. Thus, the high gene density portion of the HLA complex is extended to more than 3 Mb.

Animals↗
PubMed1993

Identification in the HLA class I region of a gene expressed late in keratinocyte differentiation.

A gene designated S has been identified in the class I region of the human major histocompatibility complex. The S gene is located 160 kb telomeric of HLA-C. It is expressed at high levels as 2.2-kb and 2.6-kb mRNAs in human skin. No homologous transcripts were detected in other tissues including placenta, liver, spleen, thymus, and brain. In situ hybridization showed that S gene expression was restricted to the differentiating keratinocytes in the granular layer of the epidermis. The predicted amino acid sequence of the S protein was remarkable for its high content of serine, glycine, and proline. There were significant similarities with the amino acid sequences of loricrin, keratin 1, and keratin 10, all major components of the granular-cell layer. The selective expression of the S gene in the granular-cell layer in the epidermis suggests a role in the developmental program of differentiating keratinocytes. Furthermore, in light of the recognized association of psoriasis vulgaris, a disorder of keratinocyte proliferation, with alleles of HLA-C, this gene may contribute primarily to the pathogenesis of this common disorder.

Amino Acid Sequence↗
PubMed1993

Simultaneous transfer of four functional genes from the HLA class II region into mammalian cells by fusion with yeast spheroplasts carrying an artificial chromosome.

Gene transfer using yeast artificial chromosome (YAC) clones provides an opportunity to study the expression of several linked genes within an environment more closely approximating their normal chromosomal context. A YAC clone spanning 330 kb of the HLA class II region from centromeric of TAP 1 to telomeric of HLA-DQA1 was retrofitted by homologous recombination with a neomycin plasmid targeted either to an Alu repeat sequence within the YAC genomic insert or to the Ura-3 gene within the right arm of the YAC vector. The modified YAC clones were transferred to Chinese hamster ovary or L cells by spheroplast fusion. Eight of 14 Alu-retrofitted and 10 of 15 right arm-retrofitted neomycin clones retained the six human loci known to be encoded by the YAC as well as portions of the left and right YAC vector arms. All tested L cell transformants showed IFN-gamma-inducible TAP 1 and TAP 2 mRNA expression. Two of eight analyzed clones expressed HLA-DQB mRNA and one of four expressed HLA-DQA. Cells expressing both the HLA-DQA and -DQB mRNA showed HLA-DQ cell surface expression. These studies establish the feasibility of introducing groups of functional genes into mammalian cells by spheroplast fusion with a single YAC clone.

Animals↗
PubMed1993

Highly informative typing of the human TNF locus using six adjacent polymorphic markers.

The human tumor necrosis factor locus (TNF locus) is located within the major histocompatibility complex between the class III genes and HLA-B. We recently characterized and studied two closely linked highly informative dinucleotide repeats (AC/GT)n (designated TNFa) and (TC/GA)k (designated TNFb) in the upstream region of the human TNF-beta (lymphotoxin) gene. We also characterized two linked (TC/GA) and (TC/GA)-like repeats located downstream of the TNF-alpha gene, designated TNFe and TNFd, respectively. Here, we combine these four markers together with a biallelic TC/GA repeat in the first intron of the TNF-beta gene (TNFc) and a biallelic NcoI RFLP (TNFn) to type 105 cell lines from the American Society of Histocompatibility and Immunogenetics Workshop and the Center for Human Polymorphism Studies reference panels of HLA typing cell lines. These 6 polymorphic markers define 35 distinct TNF haplotypes and together with the reference panel can be used for disease association and population genetics studies.

Alleles↗
PubMed1993

Chromosomal localization of five murine HSP70 gene family members: Hsp70-1, Hsp70-2, Hsp70-3, Hsc70t, and Grp78.

The 70,000-D heat shock protein (HSP70) gene family includes both heat-inducible and constitutively expressed genes. We have mapped five murine HSP70 genes to specific sites on three separate chromosomes. Southern blot analysis of Chinese hamster x mouse somatic cell DNAs was used to assign the gene for the 78,000-D glucose-regulated protein (Grp78) to Chromosome (Chr) 2, the male germ cell-specific Hsp70-2 and Hsc70t genes to Chr 12 and Chr 17, respectively, and the heat-inducible Hsp70-3 gene also to Chr 17. Southern blot analysis of DNA from the progeny of two multilocus crosses confirmed the Grp78 location on Chr 2 and suggested the order: centromere-Vim-Abl-Grp78-Hc. Similar analysis also confirmed the initial Hsp70-2 assignment to Chr 12 with the order: Hsp70-2-Aat-Igh. The Hsp70-3 and Hsc70t genes on Chr 17, along with the heat-inducible Hsp70-1 gene, were further localized by Southern blot analysis of genomic clones to the H-2 histocompatibility region with the order: Hsp70-1-Hsp70-3-Hsc70t-Bat-6 (human G7a, valyl-tRNA synthetase).

Animals↗
PubMed1993

Regulation of IL-1 gene expression: differential responsiveness of murine macrophage lines.

In order to begin to define the mechanisms by which lipopolysaccharide (LPS) regulates IL-1 gene expression, we have examined IL-1 RNA levels, the transcription rate of the IL-1 genes, and IL-1 mRNA stabilities in P388D1/C, RAW264.7, and murine peritoneal exudate cells (PEC). These experiments showed that total cellular IL-1 RNA levels and IL-1 transcription rates were dramatically upregulated in all three cell types. In all cases, IL-1 alpha and IL-1 beta cellular RNA levels and gene transcription rates were regulated in parallel. However, the profiles of IL-1 gene activation during the 24 h after LPS treatment differed in these three cell types. Additionally, culture in the presence of actinomycin D (Act D) showed differential stabilities of the IL-1 alpha and IL-1 beta RNAs in these cells. In peritoneal exudate cells, the half-lives (t1/2) of the IL-1 alpha and IL-1 beta RNAs were each > 8 h. In RAW264.7 cells, the stability of the IL-1 beta RNA was greater than the IL-1 alpha RNA (t1/2 > 8 h and approximately 6 h, respectively). In P388D1/C cells, the t1/2's of the IL-1 alpha and beta RNAs varied depending on the time of addition of actinomycin D. This and other data suggest that components of the IL-1 RNA catabolic pathway are labile and sensitive to treatment with actinomycin D. Together these data indicate that the two IL-1 genes show a diverse regulatory repertoire, even within related mononuclear phagocytic cells.

Animals↗
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↗