A polymorphic microsatellite in the tumor necrosis factor alpha promoter identifies an allele unique to the NZW mouse strain.

We have amplified a (CA)n:(GT)n microsatellite from the TNF promoters of a panel of mouse strains using the polymerase chain reaction. The length of the microsatellites was polymorphic, with eight alleles observed among 15 inbred strains bearing seven distinct H-2 haplotypes, and four outbred strains. In B10 congenic strains, the TNF allele detected by microsatellite polymorphism segregated with the MHC, and in recombinant haplotypes (NOD, NZW), it segregated with H-2D. The TNF allele found in the NZW strain (H-2z) was distinct from those of all other haplotypes, consistent with the hypothesis that this strain may carry a genetic defect in TNF production.

Multiple specificities in the repertoire of a melanoma patient's cytolytic T lymphocytes directed against tumor antigen MAGE-1.A1.

Peptide MAGE-1.A1 is a nonamer derived from protein MAGE-1 that can associate with the HLA-A1 molecule. It was shown previously to be recognized by an antitumor cytolytic T lymphocyte (CTL) clone derived from the blood of melanoma patient MZ2. We derived two other anti-MAGE-1.A1 CTL clones from different blood samples of the same patient and compared the fine specificity of recognition of the three CTL by testing them on variant MAGE-1.A1 peptides incorporating different amino acid substitutions. The epitopes recognized by the CTL proved to be different. While modifications of residues at positions 5, 6, or 7 in the antigenic peptide affected recognition by the three CTL, each of the modifications of residues at positions 1, 4, or 8 affected recognition by one CTL only. The sequences of both the alpha and beta chains of the T cell antigen receptor of the three CTL were completely different. The results indicate a long-lasting diversity in terms of fine specificity and of T cell antigen receptor structure in the repertoire of antitumor CTL derived from the blood of a melanoma patient and directed against a defined tumor antigen.

Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages.

We have explored the cis-acting elements necessary for the LPS-mediated activation of the mouse TNF-alpha promoter by transfecting a set of 5' deletion mutants linked to the CAT reporter gene into primary bone marrow-derived macrophages. A major drop in inducibility by LPS was seen upon deletion of a region mapping between nt -655 and nt -451. Gel retardation assays revealed that LPS induced the appearance in this region of several specific DNA-protein complexes mapping to sequence motifs with strong homology to the kappa B enhancer. Constructs containing two or more copies of one of the kappa B enhancer motifs linked to a heterologous promoter were inducible by LPS. Additional deletion of a region between nt -301 and nt -241, which contains a MHC class II-like "Y box" and formed a Y box-specific complex with a protein whose concentration was increased by LPS, caused a nearly complete loss of inducibility by LPS. We speculate that NF-kappa B and/or related proteins are involved in the LPS-induced transcriptional activation of the TNF-alpha gene, and that factors interacting with the Y box can additionally modulate the activity of the gene in macrophages.

Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase.

We purified CALLA from human kidney and isolated a cDNA clone reactive with two oligonucleotide probes corresponding to two distinct peptides. The amino acid sequence translated from the CALLA cDNA revealed 100% identity with that of human neutral endopeptidase (NEP, enkephalinase). The distribution of CALLA antigen and NEP in normal tissues are similar.

Analysis of tumor necrosis factor promoter responses to ultraviolet light.

Ultraviolet (UV) light induces the biosynthesis of chloramphenicol acetyltransferase (CAT) in the skin of mice bearing the CATTNF reporter transgene. Moreover, nuclear run-on assays indicate that UV light induces transcription of the TNF gene in RAW 264.7 macrophages. These observations suggest that the TNF gene (and/or its mRNA product) responds to signals elicited by UV light. To identify transcriptional UV response elements within the TNF promoter, and to determine whether a posttranscriptional response might also exist, a series of reporter constructs using a CAT coding sequence attached to various portions of the TNF promoter and 3' untranslated region were devised and transfected into several cultured cell lines. All cells tested were found to be UV responsive, and in NIH 3T3 cells, induction was found to depend upon two general regions of the promoter. The more distal region encompassed nucleotides (nt) -1059 through -451 with respect to the cap site, while the more proximal region spanned nt -403 through -261. A negative element, blocking the UV response, was interposed (nt -451 through -403). As with the response to LPS, the response to UV irradiation appears to involve translational activation in macrophages. However, the UV and LPS signaling pathways have little in common with one another, as indicated by three observations. First, no difference in responsiveness was observed on comparison of TNF gene induction in macrophages derived from C3H/HeN as opposed to C3H/HeJ mice. Second, cell fusion studies showed that while the LPS signaling pathway is extinguished by fusion of RAW 264.7 cells with NIH 3T3 cells, the UV signaling pathway remained intact. Finally, induction did not depend upon the NF-kappa B binding sites that are known to be required for LPS response in macrophages, since mutation of these sites did not impair the UV response.

Common acute lymphoblastic leukemia antigen expressed on leukemia and melanoma cell lines has neutral endopeptidase activity.

We have previously reported that the amino acid sequence of the common acute lymphoblastic leukemia antigen (CALLA, CD10) translated from a normal human kidney cDNA clone is identical to that of neutral endopeptidase (NEP, EC 3.4.24.11). In this study, we show that by flow cytometry, a monoclonal antibody (135A3) produced against rabbit NEP reacted selectively with leukemia and melanoma cell lines expressing CALLA on their surface. A glycoprotein of apparent Mr 100,000 was immunoprecipitated from surface labeled NALM-1 leukemia or Mel-1477 melanoma cells with monoclonal antibodies to NEP (135A3) or CALLA (44C10). mRNAs hybridizing to a NEP-specific probe were present in CALLA+ leukemia and melanoma cell lines, but absent from CALLA- lines. NEP enzymatic activity was detected on intact cells from CALLA+ lines, but not CALLA- lines. The activity was blocked by two selective inhibitors of NEP, thiorphan and phosphoramidon. CALLA antigen purified from the NALM-6 leukemic cell line by affinity to 44C10-IgG Sepharose retained a peptidase activity that was completely blocked by thiorphan and phosphoramidon. Thus the CALLA antigen present at the surface of leukemia and melanoma cell lines is an enzymatically active neutral endopeptidase.

Modeling sequencing errors by combining Hidden Markov models.

Among the largest resources for biological sequence data is the large amount of expressed sequence tags (ESTs) available in public and proprietary databases. ESTs provide information on transcripts but for technical reasons they often contain sequencing errors. Therefore, when analyzing EST sequences computationally, such errors must be taken into account. Earlier attempts to model error prone coding regions have shown good performance in detecting and predicting these while correcting sequencing errors using codon usage frequencies. In the research presented here, we improve the detection of translation start and stop sites by integrating a more complex mRNA model with codon usage bias based error correction into one hidden Markov model (HMM), thus generalizing this error correction approach to more complex HMMs. We show that our method maintains the performance in detecting coding sequences.

Cytokine regulation of TNF-alpha mRNA and protein production by unprimed macrophages from C57Bl/6 and NZW mice.

It is well known that bacterial lipopolysaccharide (LPS) induces the synthesis of tumor necrosis factor alpha (TNF-alpha) and other inflammatory cytokines by primary monocytes and macrophages and that the Th1 lymphokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), augment this response. We investigated the ability of IL-2 and IFN-gamma to induce the production of TNF-alpha mRNA and protein independently of LPS and the modulation of this response by macrophage colony-stimulating factor (M-CSF) and IL-10. We found that IL-2 and IFN-gamma were both able to induce the accumulation of TNF-alpha mRNA, albeit with slower kinetics than LPS, and that they acted synergistically. However, very little TNF bioactivity was secreted by lymphokine-stimulated macrophages unless LPS was also added. This finding underscores the importance of translational effects in the control of TNF production. M-CSF and IL-10 strongly inhibited TNF production at the level of both mRNA and bioactivity but had no effect on the production of IL-6. Bone marrow-derived or thioglycollate-elicited macrophages from the NZW mouse strain, which have been reported to be deficient in their ability to produce TNF, were at least as responsive to LPS or lymphokines as those taken from the C57Bl/6 strain and were similarly affected by M-CSF and IL-10. Therefore, the genetic defect of NZW mice is not a primary deficiency in TNF production.

Survival-independent function of NF-kappaB/Rel during late stages of thymocyte differentiation.

Transcription factors of the NF-kappaB/Rel family are important mediators of extracellular signals. Their implication in positive selection of thymocytes is suggested by a defective thymic development in transgenic mice that over-express IkappaB in thymocytes. These mice exhibit an accumulation of an unusually prominent population of TCRhigh/CD4/CD8 double positive cells in the thymus and a dramatic reduction of CD4+ and CD8+ cells in the periphery. The present study addresses the role of NF-kappaB in survival and differentiation processes of maturing thymocytes using IkappaB/bcl-2 and IkappaB/HY double-transgenic mice. Neither the introduction of the anti-apoptosis gene bcl-2 nor the positively selecting background in female HY transgenic mice resulted in a rescue of the maturational defects observed in the thymus of IkappaB transgenic mice. Thus, rather than promoting survival the main role of NF-kappaB/Rel proteins during positive selection of thymocytes appears to be the mediation of differentiation signals.

Structural analysis of the rabbit TNF locus, containing the genes encoding TNF-beta (lymphotoxin) and TNF-alpha (tumor necrosis factor).

The genes coding for 20-kDa lymphotoxin (TNF-beta) and tumor necrosis factor (TNF-alpha) have been cloned from a rabbit genomic library. The two genes are tandemly arranged and separated by only 1 kb of DNA, as previously observed in human and mouse genomes. We have sequenced the entire rabbit lymphotoxin-encoding gene and inferred the primary structure of rabbit TNF-beta, whose cDNA is not yet cloned. We also analysed the upstream sequences of the rabbit TNF-beta and TNF-alpha genes and identified a number of potential binding sites for known nuclear transcription factors, and in particular several putative kappa B-type sequences.

Bcl-2 protects from oxidative damage and apoptotic cell death without interfering with activation of NF-kappa B by TNF.

The recent demonstration of the anti-oxidant properties of the Bcl-2 gene product suggested that expression of Bcl-2 may interfere with the nuclear migration of the NF-kappa B transcription factor, which is thought to depend on the presence of reactive oxygen intermediates. In mouse L cells, overexpression of Bcl-2 interfered with the activation of NF-kappa B by H2O2. However, Bcl-2 had no effect on the activation of NF-kappa B by TNF, even though it protected cells from TNF-induced apoptosis. The effects of exogenous pyrrolidine dithiocarbamate were very similar to those of Bcl-2 overexpression. We conclude that the protective effects of anti-oxidants against induced apoptotic cell death are unrelated to their ability to interfere with NF-kappa B activation.

A unique signature identifies a family of zinc-dependent metallopeptidases.

The primary sequence motif HExxH has been found in many zinc-dependent endopeptidases. We show that a larger signature comprising this sequence is common to most of the known zinc-dependent endopeptidases, and that the presence of the signature can be indicative of membership in the family. A search of the protein sequence databases for entries containing the signature retrieved several unexpected potential zinc endopeptidases.

Nuclear migration of NF-kappa B correlates with TNF-alpha mRNA accumulation.

Previous work on the transcriptional regulation of the mouse TNF-alpha promoter had indicated a major role for the NF-kappa B transcription factor in the induction of the gene by endotoxin. However, similar studies using the human promoter failed to establish a role for this factor. We measured the nuclear migration of NF-kappa B and the accumulation of TNF-alpha mRNA in murine T lymphoblasts and bone marrow-derived macrophages (BMDM) under different activation conditions, seeking to establish a correlation. Activation of NF-kappa B and accumulation of TNF-alpha mRNA correlated semiquantitatively under the following conditions: (1) inhibition of NF-kappa B by dithiocarbamates; (2) induction of TNF synthesis by taxol; (3) partial induction of TNF-alpha mRNA by various inducers in macrophages from lpsd mice; and (4) inhibition of NF-kappa B activation by a protease inhibitor. However, inhibition of TNF-alpha mRNA accumulation by cAMP inducers had no effect on NF-kappa B induction. We conclude that NF-kappa B translocation is necessary, but not sufficient for the transcriptional induction of the TNF-alpha gene by LPS in macrophages or by phorbol ester and ionomycin in T lymphocytes.

Reactivity of murine and human recombinant LPS-binding protein (LBP) within LPS and gram negative bacteria.

The serum lipopolysaccharide (LPS) binding protein, LBP, has been shown to greatly enhance cellular responses to low concentrations of LPS. Purified LBP facilitates the transfer of LPS to membrane-bound or soluble CD14; the CD14/LPS complex then triggers a signal in responsive cells. We have cloned and sequenced a cDNA encoding murine LBP, and produced recombinant murine LBP using a baculovirus expression system. Using either a solid-phase or a cytofluorometric assay, recombinant murine and human LBP were found to bind avidly to free LPS, but only weakly to live bacteria from most LPS-containing Gram negative strains. Binding correlated loosely with the length and composition of the polysaccharide O chains. However, recombinant LBP did bind well to all heat-killed bacterial preparations. These findings suggest that LBP could be implicated in the response to killed but not live Gram negative bacteria.

Searching the expressed sequence tag (EST) databases: panning for genes.

The genomes of living organisms contain many elements, including genes coding for proteins. The portions of the genes expressed as mature mRNA, collectively known as the transcriptome, represent only a small part of the genome. The expressed sequence tag (EST) databases contain an increasingly large part of the transcriptome of many species. For this reason, these databases are probably the most abundant source of new coding sequences available today. However, the raw data deposited in the EST databases are to a large extent unorganised, unannotated, redundant and of relatively low quality. This paper reviews some of the characteristics of the EST data, and the methods that can be used to find novel protein sequences within them. It also documents a collection of databases, software and web sites that can be useful to biologists interested in mining the EST databases over the Internet, or in establishing a local environment for such analyses.

Structure of replicating herpes simplex virus DNA.

We have investigated the molecular anatomy of the herpes simplex virus replicative intermediates by cleavage with the restriction endonuclease BglII. We find that in populations of multiply infected cells, pulse-labeled replicating herpes simplex virus DNA contains at least two and probably all four sequence isomers. Also, it contains no detectable termini. In pulse-chase experiments, we show that endless replicative intermediates are the precursors to virion DNA and that maturation is a relatively slow process. The results are discussed in terms of their significance to possible models of herpes simplex virus DNA replication.