Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of interleukin-1alpha (IL-1alpha). To analyze the mechanisms that lead to the expression of IL-1alpha in HTLV-I-infected cell lines, we studied regulatory regions of the human IL-1alpha promoter involved in activation of the IL-1alpha gene. IL-1alpha promoter constructs drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells, which constitutively produce IL-1alpha. In a cotransfection assay, the Tax protein of both HTLV-I and HTLV-II specifically activated transcription from the IL-1alpha promoter in an uninfected Jurkat cell line. A mutant Tax protein deficient in transactivation of genes by the nuclear factor (NF)-kappaB pathway was unable to induce transcriptional activity of IL-1alpha promoter-CAT constructs, but was rescued by exogenous provision of p65/p50 NF-kappaB. We found that two IL-1alpha kappaB-like sites (positions -1,065 to -1,056 and +646 to +655) specifically formed a complex with NF-kappaB-containing nuclear extract from MT-2 cells and that NF-kappaB bound with higher affinity to the 3' NF-kappaB binding site than to the 5' NF-kappaB site. Moreover, deletion of either 5' or 3' NF-kappaB sites reduced IL-1alpha promoter activity in MT-2 cells and transactivation of the IL-1alpha promoter by exogenous NF-kappaB and Tax in Jurkat cells. These data suggest a general role for Tax induction of IL-1alpha gene transcription by the NF-kappaB pathway. Expression of IL-1alpha by HTLV-I productively infected cells may be important in the hypercalcemia, osteolytic bone lesions, neutrophilia, elevation of C-reactive protein, and fever frequently seen in patients with HTLV-I-induced adult T-cell leukemia/lymphoma.
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PMID:Transactivation of the interleukin-1alpha promoter by human T-cell leukemia virus type I and type II Tax proteins. 860 59

It is thought that interference during human immunodeficiency virus type 1 (HIV-1) infection is established by downmodulation of the principal virus receptor, CD4. Here we present evidence to the contrary. At various times after primary infection, we superinfected T cells in vitro by exposure to a genetically distinct viral clone or to a virus carrying the chloramphenicol acetyltransferase gene. Replication of each virus strain was determined by restriction enzyme analysis of total cellular DNA, by PCR amplification of viral DNA, or by assay of cell extracts for chloramphenicol acetyltransferase activity. We found that efficient viral interference is established within 24 h of infection at a multiplicity of infection of 1. At that time, expression of viral structural proteins was low and infected cells displayed undiminished levels of surface CD4 and were fully susceptible to virus binding and fusion. Superinfection by either cell-free HIV-1 or cocultivation was blocked. Cells resistant to superinfection by HIV-1 remained susceptible to Moloney murine leukemia and vaccinia viruses. No interference was observed 4 h after primary infection or in cells infected with either UV-inactivated HIV-1 or a mutant virus defective in virus-cell fusion activity, indicating that binding of primary virus to CD4 is insufficient to prevent superinfection. The minimum viral requirements for this interference are that HIV-1 must be able to enter cells and synthesize viral DNA; Tat-mediated transcription is dispensable. Our results support the existence of a novel pathway to interference to HIV-1 infection, which we term postentry interference, which blocks superinfection during intracellular phases of the virus life cycle.
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PMID:Interference to human immunodeficiency virus type 1 infection in the absence of downmodulation of the principal virus receptor, CD4. 864 18

Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between transcriptional repression by FVIII cDNA elements and the stimulatory effect of butyrate on FVIII cDNA expression.
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PMID:The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate. 875 27

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.
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PMID:Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. 878 Nov 19

We compared the efficiency of human immunodeficiency virus (HIV-1) vectors that express a marker gene (chloramphenicol acetyltransferase, CAT) using different promoter elements. In one vector, CAT was expressed under the control of an internal murine leukemia virus (MuLV) long terminal repeat (LTR). In other vectors, CAT production was regulated by the HIV-1 LTR; these vectors also contained the HIV-1 tat gene and pol sequences reported to exert cis-acting positive effects on reverse transcription or gene expression. Vectors employing the Tat-driven HIV-1 LTR exhibited up to 500-fold greater CAT expression in Jurkat lymphocytes or human peripheral blood mononuclear cells compared with vectors using the internal MuLV LTR element as a promoter. This difference was not due to improved packaging of the vector RNA into virions, but to an improved level of gene expression in the target cells. Target cell CAT expression was two- to threefold higher for the vector containing the pol sequences and was only slightly less than that seen for a trans-complemented envdeleted provirus. These results indicate that defective HIV-1 vectors with efficiencies of gene transfer and expression comparable with that of HIV-1 itself are feasible.
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PMID:Use of cis- and trans-acting viral regulatory sequences to improve expression of human immunodeficiency virus vectors in human lymphocytes. 880 25

The ability of glucocorticoids (GCs) to induce death in lymphoid-origin cells is the basis for their frequent use in the therapy of various human hematological malignancies. However, the occurrence of primary or secondary GC resistance limits their clinical usefulness. Prior investigations into the mechanism of GC resistance in established human leukemic cell lines revealed loss-of-function mutations in the GC receptor (GR) gene. In this study, we analyzed the GC-resistant human acute T-cell leukemia line CEM-C1, which has been reported to express biochemically functional GR and, thus, was thought to owe its GC resistance to signal transduction changes distal from the GR. Radioligand binding assays revealed a 2-3-fold lower expression of GR in CEM-C1 than in the GC-sensitive sister cell line CEM-C7H2. Analysis of transcriptional activity using mouse mammary tumor virus-long terminal repeat-controlled chloramphenicol acetyltransferase expression in transient transfection assays confirmed the expression of functional GR in CEM-C1 but at levels lower than those in CEM-C7H2 cells. Upon molecular analyses of the GR gene and its transcripts, we found that CEM-C1 cells were heterozygous for the ligand binding domain L753F point mutation in exon 9, which is also present in GC-sensitive CEM-C7H2. No mutations, however, were found on the second GR allele of CEM-C1. To test the possibility that resistance in CEM-C1 cells might be caused by insufficient expression of GR, we established several cell lines stably transfected with rat GR expression vectors. These cell lines differed in exogenous GR expression as determined by Northern blotting and radioligand binding assays. The GR expression level in individual lines correlated well with their sensitivity to GC-induced apoptosis. Thus, GC resistance of CEM-C1 cells might be due to subthreshold expression of functional GR rather than defects in signal transduction pathways distal from the GR. Since several clinical investigations showed a correlation between reduced GR expression and poor response to GC-containing treatment, the CEM-C1 line may represent a valid model for GC resistance in human acute T-cell leukemia.
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PMID:Resistance to glucocorticoid-induced apoptosis in human T-cell acute lymphoblastic leukemia CEM-C1 cells is due to insufficient glucocorticoid receptor expression. 889 60

Leukemia-inhibitory factor (LIF) is a neuropoietin able to regulate the differentiation and the survival of many cell types, which include some neuronal populations. The present study describes the genetic construction, expression, purification and properties of a diphtheria-toxin-related LIF gene fusion in which the native receptor-binding domain of diphtheria toxin was replaced with a gene encoding human LIF. The fusion protein expressed from the chimeric tox gene was designated DT-(1-389)-LIF-(2-184)-peptide. This fusion protein has a deduced molecular mass of 65980 Da and is formed by fusion of the first 389 amino acids of diphtheria toxin to amino acids 2-184 of mature human LIF, using a linker of 34 amino acids that includes six consecutive histidine residues. The latter span allows for single-step purification of the fusion protein by Ni(2+)-resin affinity chromatography. This linker provides a high degree of flexibility between the diphtheria toxin and LIF domains, thereby permitting aggregation-free refolding of the chimeric protein while bound to the affinity column. Both LIF and DT-(1-389)-LIF-(2-184)-peptide induced the phosphorylation of CLIP1 and CLIP2 in LIF-responsive neuroblastoma SH-N-BE cells. DT-(1-389)-LIF-(2-184)-peptide was selectively cytotoxic for cultured neuroblastoma cells bearing the LIF receptor, and for sympathetic neurons. The cytotoxic action of DT-(1-389)-LIF-(2-184)-peptide, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment, and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The latter point was confirmed by the fact that, while both LIF and DT-(1-389)-LIF-(2-184)-peptide increased c-fos mRNA expression in SH-N-BE cells, only LIF induced proenkephalin and c-fos promoter activities in cells transiently transfected with c-fos-chloramphenicol acetyltransferase and proenkephalin-chloramphenicol acetyltransferase fusion genes. Mutational analysis suggested that the C-terminal helix (helix D) of human LIF may, in part, constitute or contribute to the active site for LIF receptor binding and cell activation. The cytotoxic properties of DT-(1-389)-LIF-(2-184)-peptide may be useful in selectively depleting neuronal and immune cell populations that express the LIF beta receptor.
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PMID:Synthesis, cytotoxic properties and effects on early and late gene induction of a chimeric diphtheria toxin-leukemia-inhibitory factor protein. 891 49

Adult T cell leukemia/lymphoma (ATL) is derived from CD4+ T cells and has a poor prognosis because of its resistance to chemotherapy. To evaluate the effectiveness of gene therapy for ATL, the effect of ganciclovir on ATL cell lines transfected with the thymidine kinase gene of herpes simplex virus type 1 (HSV-TK) was analyzed. To transfer the HSV-TK gene to ATL cells, a human immunodeficiency virus (HIV) vector that has specific infectivity to CD4+ cells was used. HSV-TK was inserted into the long terminal repeats of HIV-1 and driven by the SL3 promoter HXBSL3TK. HXBSL3TK was co-transfected with HXBCAT as a reporter into MT2 or HUT102 cells by DEAE-dextran. The cells were incubated with ganciclovir, and chloramphenicol acetyltransferase (CAT) activity was analyzed. The CAT activity of the MT2 cells and HUT102 cells transfected with HXBSL3TK decreased dose-dependently with ganciclovir. HXBSL3TK was also co-transfected into COS cells with an HIV-1 packaging vector that has gag, pol, and env driven by a cytomegalovirus promoter. The supernatant was transferred to MT2 cells or Raji cells and incubated with ganciclovir. Ninety percent of the MT2 cells transduced by HXBSL3TK and incubated with ganciclovir were killed, but Raji cells were not killed. In addition, HXBTK that expresses the HSV-TK gene and Tat gene driven by the LTR of HIV-1 was constructed. HXBTK had a higher expression of the HSV-TK gene and higher sensitivity to ganciclovir than did HXBSL3TK.
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PMID:Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1. 895 10

Previously, we showed that surface expression of intercellular adhesion molecule 1 (ICAM-1) was strongly upregulated in T cells carrying proviral human T-cell leukemia virus type 1 (HTLV-1) and that the viral transactivator protein Tax1 was capable of inducing the ICAM-1 gene. To determine the responsive elements in the human ICAM-1 gene promoter, a reporter construct in which the 5'-flanking 4.4-kb region of the ICAM-1 gene was linked to the promoterless chloramphenicol acetyltransferase (CAT) gene was cotransfected with expression vectors for Tax1 and Tax2, both of which were separately confirmed to be potent transactivators of the HTLV-1 long terminal repeat (LTR). Tax1 strongly activated the ICAM-1 promoter in all the cell lines tested: three T-cell lines (Jurkat, MOLT-4, and CEM), one monocytoid cell line (U937), and HeLa. Unexpectedly, Tax2 activated the ICAM-1 promoter only in HeLa. By deletion and mutation analyses of the 1.3-kb 5'-flanking region, we found that Tax1 transactivated the ICAM-1 promoter mainly via a cyclic AMP-responsive element (CRE)-like site at -630 to -624 in the Jurkat T-cell line and via an NF-kappaB site at -185 to -177 and an SP-1 site at -59 to -54 in HeLa. On the other hand, Tax2 was totally inactive on the ICAM-1 promoter in Jurkat but transactivated the promoter via the NF-kappaB site at -185 to -177 in HeLa. Gel mobility shift assays demonstrated proteins specifically binding to the CRE-like site at -630 to -624 in Tax1-expressing T-cell lines. Stable expression of Tax1 but not Tax2 in Jurkat subclones enhanced the surface expression of ICAM-1. The differential ability of Tax1 and Tax2 in transactivation of the ICAM-1 gene may be related to the differential pathogenicity of HTLV-1 and HTLV-2.
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PMID:Differential transactivation of the intercellular adhesion molecule 1 gene promoter by Tax1 and Tax2 of human T-cell leukemia viruses. 897 Sep 74

The infection of cells with Moloney murine leukemia virus (M-MuLV) causes an increase in specific cellular gene products, including the major histocompatibility complex (MHC) class I antigens. This upregulation occurs through a transactivation process mediated by the long terminal repeat (LTR) of M-MuLV, and we show here that the gene activation response to the LTR requires at least one specific cis element within the MHC proximal promoter region. Nested deletions of MHC class I H-2Kb gene promoter sequence were subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector and then transiently introduced into BALB/c-3T3 cells expressing M-MuLV or cotransfected into BALB/c-3T3 cells with a vector containing subgenomic portions of the virus, including the LTR. CAT activity assays demonstrated that a minimal H-2Kb gene promoter (-64 to +12) contained elements sufficient for this transactivation. DNase I footprinting assays located a protein-binding site in the region of -64 to -34 bp from the transcriptional start site, and point mutation analysis confirmed the location of this cis-acting element, designated the let response element (LRE), and defined a binding motif. This LRE is distinct from binding sites for currently known transcription factors in the class I MHC gene promoter and is conserved in the promoters of human and murine MHC class I genes. Mutation of the LRE resulted in dramatic reduction in both DNA-protein binding activity in electrophoretic mobility shift assay and in the ability of the mutated promoter to respond to retroviral transactivation. Addition of the LRE to a heterologous promoter conferred the ability to respond to retroviral transactivation.
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PMID:Identification of a cis-acting element in the class I major histocompatibility complex gene promoter responsive to activation by retroviral sequences. 899 14


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