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)

Analysis of the p50 and p65 subunits of the NF-kappa B transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known kappa B DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50- and c-Rel-selected sequences. Using either a p50- or p65-selected kappa B motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappa B complex. Similarly, in transfection experiments in which the selective kappa B binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65- and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-kappa B activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappa B complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific kappa B motifs present.
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PMID:Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation. 140 30

The human immunodeficiency virus (HIV) enhancer element is important in the regulation of HIV gene expression. A number of cellular proteins have been demonstrated to bind to the NF-kappa B motifs in this element. The genes encoding several of these proteins, including members of the rel family and PRDII-BF1, have been cloned. We characterized the binding of proteins encoded by the human c-rel and PRDII-BF1 genes to HIV NF-kappa B motifs and related enhancer elements. Both the human c-rel protein and two proteins derived from the PRDII-BF1 gene by alternative splicing bound specifically to the HIV NF-kappa B motif and related enhancer elements found in the immunoglobulin kappa, class I major histocompatibility complex, and interleukin-2 receptor genes. To determine the role of these factors in regulating HIV gene expression, we fused the cDNAs encoding either of the two proteins derived by alternative splicing of the PRDII-BF1 gene or the c-rel gene to the DNA binding region of the yeast transcription factor GAL4. GAL4 binding sites were inserted in place of the native HIV enhancer sequences in an HIV long terminal repeat chloramphenicol acetyltransferase construct. Cotransfection of these constructs revealed that c-rel was a strong activator of basal HIV gene expression but did not result in synergistic effects in the presence of tat. PRDII-BF1-derived cDNAs did not result in stimulation of either basal or tat-induced activated gene expression. These results indicate that multiple enhancer binding proteins may potentially regulate HIV in both a positive and negative manner.
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PMID:Regulation of human immunodeficiency virus enhancer function by PRDII-BF1 and c-rel gene products. 172 88

Our studies originally demonstrated that the v-rel oncoprotein repressed gene expression in chicken lymphoid cells, while it activated transcription in rodent fibroblasts. Here we report that the c-rel protein can activate expression of genes linked to kappa B motifs when low levels of endogenous kappa B-binding activity are present. In contrast v-rel, and to a lesser extent c-rel, inhibit NF-kappa B-mediated activation of the human immunodeficiency virus long terminal repeat (HIV LTR) in phorbol ester-stimulated HeLa cells. Competition assays show that v-rel competitively inhibits both NF-kappa B and c-rel-mediated transcriptional activation. Analysis of mutant HIV LTR-chloramphenicol acetyltransferase (CAT) constructs in which all Sp1 or both NF-kappa B elements have been deleted shows that NF-kappa B motifs are required for rel-mediated effects on gene expression. Transforming v-rel mutants compete efficiently with phorbol ester-activated kappa B factors, whereas a transformation-defective mutant of v-rel is impaired in this activity. Taken together, these results strengthen the hypothesis that v-rel functions as a dominant interfering member of rel family proteins. These results also suggest that the ability of v- and c-rel to activate or repress gene expression in specific cells may result from their capacity to compete with endogenous rel family proteins whose expression and/or activity are cell-specific.
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PMID:Transcriptional activity of rel family proteins. 174 Nov 61

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.
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PMID:Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3. 747 23

Expression of the c-myb proto-oncogene is primarily detected in normal tissue and tumor cell lines of immature hematopoietic origin, and the down-regulation of c-myb expression is associated with hematopoietic maturation. Cell lines that represent mature, differentiated hematopoietic cell types contain 10-100-fold less c-myb mRNA than immature hematopoietic cell types. Differences in steady-state c-myb mRNA levels appear to be primarily maintained by a conditional block to transcription elongation that occurs in the first intron of the gene. The block to transcription elongation has been mapped, using nuclear run-on analysis, to a region of DNA sequence that is highly conserved between mouse and man. Two sets of DNA-protein interactions, flanking the site of the block to transcription elongation, were detected that exhibited DNA-binding activities that strongly correlated with low steady-state c-myb mRNA levels. Several criteria demonstrated that members of the nuclear factor kappa B (NF-kappa B) family of transcription factors were involved in the DNA-protein interactions identified in these two sets. Surprisingly, cotransfection experiments demonstrated that coexpression of members of the NF-kappa B family, specifically p50 with p65 and p65 with c-Rel, transactivated a c-myb/chloramphenicol acetyltransferase reporter construct that contained 5'-flanking sequences, exon I, intron I, and exon II of the c-myb gene. Transactivation by these heterodimer combinations was dependent on regions of the c-myb first intron containing the NF-kappa B-binding sites. These findings suggest that NF-kappa B family members may be involved in either modifying the efficiency of transcription attenuation or acting as an enhancer-like activity to increase transcription initiation. Thus, the regulation of c-myb transcription may be quite complex, and members of the NF-kappa B family likely play an important role in this regulation.
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PMID:Members of the nuclear factor kappa B family transactivate the murine c-myb gene. 770 14

Multiple regulatory domains within the -100 region of the beta interferon (IFN-beta) promoter control the inducible response of the IFN gene to virus infection. In this study, we demonstrate that the formation of NF-kappa B-specific complexes on the positive regulatory domain II (PRDII) precedes the onset of detectable IFN-beta transcription in Sendai virus-infected cells. By using NF-kappa B subunit-specific antibodies, a temporal shift in the composition of NF-kappa B subunits in association with the PRDII domain is detected as a function of time after virus infection. Furthermore, a virus-induced degradation of I kappa B alpha (MAD3) protein is observed between 2 and 8 h after infection; at later times, de novo synthesis of I kappa B alpha restores I kappa B alpha to levels found in uninduced cells and correlates with the down regulation of IFN-beta transcription. In cotransfection experiments using various NF-kappa B subunit expression plasmids and two copies of PRDII/NF-kappa B linked to a chloramphenicol acetyltransferase reporter gene, we demonstrate that expression of p65, c-Rel, or p50 or combinations of p50-p65 and p65-c-Rel differentially stimulated PRDII-dependent transcription. Coexpression of I kappa B alpha completely abrogated p65-, c-Rel-, or p65-p50-induced gene activity. When the entire IFN-beta promoter (-281 to +19) was used in coexpression studies, synergistic stimulation of IFN-beta promoter activity was obtained when NF-kappa B subunits were coexpressed together with the IFN regulatory factor 1 (IRF-1) transcription factor. Overexpression of either I kappa B or the IRF-2 repressor was able to abrogate inducibility of the IFN-beta promoter. Thus, multiple regulatory events--including differential activation of DNA-binding NF-kappa B heterodimers, degradation of I kappa B alpha, synergistic interaction between IRF-1 and NF-kappa B, and decreased repression by I kappa B and IRF-2--are all required for the transcriptional activation of the IFN-beta promoter.
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PMID:Viral induction of the human beta interferon promoter: modulation of transcription by NF-kappa B/rel proteins and interferon regulatory factors. 803 74

Infection-induced activation of the human cytomegalovirus major immediate early enhancer/promoter has been shown to be regulated primarily by transcription factor NF-kappa B cis elements. However, the mechanism(s) by which human cytomegalovirus induces NF-kappa B activity is unknown. A study was therefore undertaken to determine how this virus would affect normal NF-kappa B regulation. Viral infection of fibroblasts resulted in the specific stimulation of promoters containing major histocompatibility complex NF-kappa B cis elements fused upstream of the chloramphenicol acetyltransferase reporter gene. Electrophoretic mobility shift assays of nuclear extracts derived from mock- and virus-infected cells showed dramatic and sustained increases in DNA-binding proteins specific for these NF-kappa B sequences. Experiments using MAD-3 I kappa B, a specific inhibitor of NF-kappa B, and antibodies directed against rel family members demonstrated that the induced binding activities contained p50 and p65 proteins but not c-rel. Northern analysis indicated maximal levels of p50 mRNA by 4 h postinfection, whereas p65 and MAD-3 I kappa B mRNA accumulation peaked at 48-72 h postinfection, suggesting different regulatory mechanisms for p50 and p65/I kappa B genes. Electrophoretic mobility shift assays with deoxycholate-treated cytoplasmic extracts demonstrated a 3- to 4-fold decrease in the cytosolic stores of NF-kappa B binding activity by 4 h postinfection. Western blots probed with antibodies directed against MAD-3 I kappa B or pp40 (a protein isolated from chicken with sequence and biochemical properties similar to those of MAD-3 I kappa B) indicated that a cross-reactive peptide of 39 kDa was no longer detectable after 24 h postinfection. These results demonstrate that the activation and maintenance of nuclear NF-kappa B DNA binding and enhancer activities upon human cytomegalovirus infection occurs by multiple mechanisms.
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PMID:Multiple mechanisms are implicated in the regulation of NF-kappa B activity during human cytomegalovirus infection. 838 32

Optimal activation of T cells requires at least two signals. One signal can be delivered by the antigen-specific T-cell receptor, and the second signal is provided by the costimulatory molecule(s) delivered by the antigen-presenting cell. CD28 is a T-cell surface molecule and stimulation through this protein plays an important role in delivering the second activation signal. In this report, we show that in human peripheral blood T cells, CD28-mediated signal transduction involves the rel family proteins--c-Rel, p50, and p65. Treatment of peripheral blood T cells with phorbol 12-myristate 13-acetate (PMA) and anti-CD28 monoclonal antibody (mAb) results in augmentation of nuclear c-Rel, p50, and p65, and this augmentation can occur in the presence of the immunosuppressant cyclosporin A. It is also shown in this report that, in response to PMA/anti-CD28 mAb or anti-CD3/anti-CD28 mAb, c-Rel, p50, and p65 are associated with CD28-responsive element present in the promoter of the human interleukin 2 gene. The functional significance of c-Rel involvement in the CD28-responsive complex is demonstrated by transient transfection analysis, where cotransfection of c-Rel augments the level of expression of a chloramphenicol acetyltransferase reporter gene linked to the CD28-responsive element.
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PMID:The interleukin 2 CD28-responsive complex contains at least three members of the NF kappa B family: c-Rel, p50, and p65. 838 23

The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.
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PMID:Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop. 841 23

The lysozyme gene is expressed at a low level in myeloblasts and is progressively activated to constitutively high expression in mature macrophages. The binding activity of the newly defined NF-kappa B/Rel family of transcription factors increases during the terminal differentiation of macrophages. In this study, I show that NF-kappa B/Rel-like proteins bind to the nuclear factor kappa B (kappa B)-like sequence of the lysozyme promoter. These binding activities were induced by treatment of HD11 cells with lipopolysaccharide. Immunomobility shift assays show that c-Rel is possibly a factor in the complexes that bind to the kappa B-like sequence lys kappa B. Binding activity to one of the protein complexes seems to be regulated by phosphorylation. In fact, overexpression of p65 and c-Rel stimulates expression of the chloramphenicol acetyltransferase gene controlled by the lysozyme promoter. Furthermore, co-transfection experiments reveal that the kappa B-like sequence within the lysozyme promoter mediates the transactivation by p65 and c-Rel. These results indicate that the p65 and c-Rel could be components of the protein complexes that bind to the kappa B-like sequence and this binding could contribute to the progressively activated expression of the lysozyme gene during the terminal differentiation of macrophages.
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PMID:Transcriptional activation of the chicken lysozyme gene by NF-kappa Bp65 (RelA) and c-Rel, but not by NF-kappa Bp50. 854 7


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