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)

The relationship between transcription of alpha and beta interferon (IFN-alpha and IFN-beta) genes and the interaction of IFN promoter-binding transcription factors has been examined in monoblastoid U937 cells following priming with recombinant IFN-alpha 2 (rIFN-alpha 2) and Sendai virus induction. Pretreatment of U937 cells with rIFN-alpha 2 prior to Sendai virus infection increased the mRNA levels of IFN-alpha 1, IFN-alpha 2, and IFN-beta as well as the final yield of biologically active IFN. Analysis of nuclear protein-IFN promoter DNA interactions by electrophoretic mobility-shift assays demonstrated increased factor binding to IFN-alpha 1 and IFN-beta regulatory domains, although no new induction-specific complexes were identified. On the basis of competition electrophoretic mobility-shift assay results, factors interacting with the IFN-alpha 1 and IFN-beta promoters appear to be distinct DNA-binding proteins. U937 factor binding was localized to the P2 domain (-64 to -55) of the IFN-beta regulatory element, a sequence motif with 80% homology to the recognition site of transcription factor NF-kappa B. Protein-DNA interactions within the IFN-beta P2 domain were, in fact, specifically competed by either excess homologous P2 fragment or the human immunodeficiency virus enhancer element which contains two duplicated NF-kappa B recognition sites. Hybrid promoter-chloramphenicol acetyltransferase fusion plasmids, containing either the IFN-beta regulatory element or the human immunodeficiency virus enhancer element linked to the simian virus 40 promoter, were analyzed for virus and phorbol ester inducibility in epithelial and lymphoid cells, respectively. In the 293 cell line, both plasmids were constitutively expressed but not virus inducible, while in Jurkat cells, chloramphenicol acetyltransferase activity from these plasmids was induced by tumor-promoting agent treatment. These experiments suggest that induction of IFN gene expression may be controlled in part by transcription regulatory proteins binding to an NF-kappa B-like site within the IFN-beta promoter.
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PMID:Induction of human interferon gene expression is associated with a nuclear factor that interacts with the NF-kappa B site of the human immunodeficiency virus enhancer. 254 71

To determine the location of sites that may be important for the function of the promoter of the epidermal growth factor (EGF) receptor gene and to characterize the factors that bind to these sites, the promoter region was analyzed by deletion analysis, exonuclease III protection and gel retardation assays with crude and fractionated nuclear extracts and DNase I footprinting using purified Sp1. Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions of the EGF receptor gene promoter into CV-1 cells indicated that the region between -178 and -16 (initiator ATG is +1) is sufficient for promoter activity. Exonuclease III protection assays revealed the presence of eight specific nuclear protein binding sites in the region between -481 and -16. Gel retardation assays confirmed that multiple protein binding sites exist in this region (-481 to -16) and quantitatively agree with exonuclease III protection. DNase I footprinting using purified Sp1 showed that this transcription factor can bind to four sites (-457 to -440, -365 to -286, -214 to -200, and -110 to -84) in the EGF receptor gene promoter and therefore may play a role in its regulation.
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PMID:Epidermal growth factor receptor gene promoter. Deletion analysis and identification of nuclear protein binding sites. 283 11

The p53 is a nuclear protein that is associated with normal cellular proliferation and can cooperate with Ha-ras in causing cellular transformation in vitro. Lineage association is known to exist between p53 expression and normal lymphopoiesis, but not myelopoiesis. We studied the expression of p53 using chronic myelogenous leukemia (CML) cell lines, somatic hybrids of these cells, and leukemic cells from CML patients. Lymphoid CML lines expressed both p53 mRNA and protein. We also analyzed p53 synthesis by two B-lymphoid lines from the same CML patient; cells of one line were derived from the neoplastic clone, cells of the other were derived from the normal clone. Both synthesized equal amounts of a phosphorylated p53 protein. None of the myeloid CML lines expressed detectable p53 protein and two of four expressed negligible p53 mRNA. Two other myeloid CML lines and myeloid cells from three of four patients expressed p53 mRNA. These findings suggest that expression of the gene is not regulated normally in CML. Several approaches were pursued to explore the differential expression of p53. Southern blot analyses showed no gross alterations in the p53 gene from cells of either the expressing or the nonexpressing lines. No difference in the pattern of demethylated CpG sites was noted in the region of the p53 gene in cells from K562 (myeloid p53 nonexpressor) and in BV173 (lymphoid p53 expressor). The sites of demethylation clustered in and around the p53 promoter in both cell lines. Somatic hybrids formed between a p53 mRNA nonexpressor myeloid line (K562) and the parental p53 expressor lymphoid lines (Daudi, PUT) produced p53 mRNA and protein, suggesting that p53 is a dominantly expressed protein and that lack of expression in myeloid cells is not mediated by a trans-acting negative regulatory protein. DNA transfection experiments performed using the indicator gene chloramphenicol acetyltransferase attached to promoter sequences of p53 showed that these constructs were equally activated in BV173 (p53 expressor) and K562 (p53 mRNA nonexpressor). The mechanism of p53 regulation in CML remains unclear.
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PMID:p53 in chronic myelogenous leukemia. Study of mechanisms of differential expression. 328 Jul 26

12(R)-Hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid (12(R)-HETrE) is an arachidonic acid metabolite formed by the corneal epithelium of several species, porcine leukocytes, and human and rat epidermal cells. It is a potent, stereospecific proinflammatory and angiogenic factor and its synthesis is increased manyfold in inflamed tissues, e.g. cornea and skin. It is possible that the angiogenic activity of 12(R)-HETrE is due to a direct mitogenic effect on microvessel endothelial cells via yet to be elucidated cellular and molecular mechanisms. In the present study, we demonstrated the ability of 12(R)-HETrE to stimulate the growth of quiescent endothelial cells in a time- and concentration-dependent manner with a maximal effect at 0.1 nM. This effect was highly stereospecific since its enantiomer, 12(S)-HETrE, had no effect within the same concentration range. Northern blot analysis and transient transfection experiments with chloramphenicol acetyltransferase constructs of oncogene promoter regions demonstrated significant increases over control (0.5% fetal calf serum) in c-myc-, c-jun, and c-fos mRNA levels and expression in cells treated with 0.1 nM 12(R)-HETrE. Electrophoretic mobility shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE with specific radiolabeled oligonucleotides corresponding to known transcriptional binding sites, including AP-1, AP-2, SP1, TRE, NF kappa B, TFIID, OKT1, CREB, CTF/NF1, and GRE demonstrated a markedly rapid and specific increase in the binding activity of NF kappa B and to a lesser extent, AP-1. No significant increase was observed in the binding of other transcription factors assayed as compared to control (untreated) cells. Since the protooncogenes (c-fos, c-jun, and c-myc) are immediate early response genes that are implicated in the process of cell proliferation and differentiation, and activation of certain transcription factors, in particular NF kappa B, is associated with the immediate response of the cell to an injury, we propose that 12(R)HETrE's mitogenic and angiogenic activities are mediated, in part, via the activation of NF kappa B and expression of these protooncogenes.
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PMID:Activation of nuclear factor kappa B and oncogene expression by 12(R)-hydroxyeicosatrienoic acid, an angiogenic factor in microvessel endothelial cells. 752 72

Phosphorylation of HeLa SII (or TFIIS)-related nuclear protein p21/SIIR was demonstrated in transfected COS-1 cells. To test for a possible functional link between phosphorylation and the previously described Rous sarcoma virus (RSV) long terminal repeat (LTR) repression (Yeh, C.H., and Shatkin, A.J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11002-11006), p21/SIIR mutants were constructed and assayed for phosphorylation level and effect on RSV LTR-driven chloramphenicol acetyltransferase (CAT) reporter expression. A major phosphorylation target in p21/SIIR was localized to the Arg/Ser-rich region between amino acids 12 and 49. Deletion of this region impaired the ability of p21/SIIR to down-regulate RSV LTR promoter function. Four serine pairs, all displaying the Arg/Lys-Ser-Ser motif typical of phosphorylation sites, are present in p21/SIIR between positions 31 and 48. Conversion of these individual serine pairs to alanine resulted in decreased phosphorylation in each case. Mutation of the Ser36-Ser37 pair also diminished by severalfold the repression activity of p21/SIIR. The single tyrosine (Tyr155) in p21/SIIR was not detectably phosphorylated in transfected COS-1 cells, suggesting that the Ser36-Ser37 pair mediates Ser/Thr phosphorylation of p21/SIIR and is critical for LTR repression function.
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PMID:The Ser36-Ser37 pair in HeLa nuclear protein p21/SIIR mediates Ser/Thr phosphorylation and is essential for Rous sarcoma virus long terminal repeat repression. 759 88

The mouse apolipoprotein (apo) E gene from strain C57BL/6 was isolated from a genomic DNA library and its complete nucleotide sequence, together with 1.3 kilobase of 5' flanking DNA and 300 base pairs of the 3' flanking DNA, was determined. Regulatory sequences in the proximal 5' flanking region of the gene were identified. Using a chloramphenicol acetyltransferase transient assay system, positive and negative cis-acting sequences were mapped within 380 base pairs of the 5' flanking region of the mouse apoE gene. Two nuclear protein binding sites were identified within this region by DNase I footprinting. We have characterized one of these regions, termed mouse apoE regulatory sequence (MARS-2), which spans nucleotides -151 to -133. Gel mobility shift assays using oligonucleotides of the MARS-2 sequence having specific deletions or substitutions as probes or competitors showed that the essential sequence of MARS-2 required for nuclear protein binding consists of 16 nucleotides encompassing -151 to -136. When nuclear extracts from different cells were examined, L cells and mouse liver nuclear protein contained the highest levels of binding protein for the MARS-2 probe. This protein, termed MARS-2 binding protein, was purified from mouse liver nuclear extracts to homogeneity using gel filtration and MARS-2 oligonucleotide-specific column chromatographic procedures. The Mr = 66,000 binding protein showed a gel mobility shift band that was identical to that of crude nuclear extracts.
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PMID:Characterization of an upstream regulatory sequence and its binding protein in the mouse apolipoprotein E gene. 759 86

DNA sequence polymorphism in the genes encoding HLA class II proteins accounts for allelic diversity in antigen recognition and presentation and, thus, in the role of these cell surface glycoproteins as determinants of the scope of the T-cell repertoire. In addition, sequence polymorphism in the promoter-proximal transcriptional regulatory regions of these genes has been described, particularly for the HLA-DQB1 locus, where these differences may contribute to variation in locus- and allele-specific expression. In this study, we measured the effect of such regulatory sequence polymorphism on the expression of endogenous alleles of DQB1 in heterozygous cells. Quantitative reverse transcriptase-mediated PCR analysis showed that expression of the DQB1*0301 allele responded more rapidly to gamma interferon induction than that of DQB1*0302. We have analyzed functional effects of a prominent allelic polymorphism that consists of a TG dinucleotide present between the W and X1 consensus elements in the DQB1*0302 allele but missing in the DQB1*0301 allele. The dominant effect of this polymorphism was to introduce a variation in the spacing between the W and X1 elements of these two alleles. A secondary compensatory effect was specific for the TG dinucleotide itself, which was essential for the binding of a nuclear protein complex to the *0302 regulatory region immediately 5' of the X1 element. Derivatives of the DQB1 5' regulatory region were used to drive expression of the chloramphenicol acetyltransferase gene in transient transfections of human B-lymphoblastoid and gamma interferon-treated melanoma cell lines, demonstrating that the additional spacing between the W and X1 elements caused by the presence of the TG dinucleotide in the *0302 allele resulted in reduced expression compared with that driven by the *0301 fragment; this difference overshadowed an up-regulating effect on expression which corresponded to the binding of the TG-dependent nuclear protein complex. The presence of this polymorphism in multiple HLA-DQB1 alleles and in several species suggests selection for two alternative transcriptional regulatory mechanisms influencing expression of alleles of the same HLA locus.
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PMID:Functional effects of a natural polymorphism in the transcriptional regulatory sequence of HLA-DQB1. 765 94

The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of IL-1 beta gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and TNF-alpha-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the IL-1 beta gene by deletion analysis and chloramphenicol acetyltransferase assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the nuclear factor-IL6 (NF-IL6) binding site. Site-directed mutagenesis demonstrated that the two NF-IL-6 motifs could be independently activated by LAM, LPS, or TNF-alpha and that they acted in an orientation-independent manner. DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or TNF-alpha-stimulated THP-1 cells to the NF-IL6 motifs. We conclude that the two NF-IL6 sites mediate induction of IL-1 beta in response to the stimuli LAM, LPS, and TNF-alpha.
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PMID:Regulation of the interleukin-1 beta (IL-1 beta) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs. 768 3

The leader of the 6.0 kb human insulin-like growth factor 2 (IGF-2) mRNA, leader 3, has been reported to partially repress translation. In the regulation of this phenomenon, RNA-binding proteins may play a role. Using UV-irradiation crosslinking, we found specific binding of four proteins (57, 43, 37 and 36 kDa) to this leader. Binding of these proteins to RNA proved to be highly sensitive to the potassium chloride concentration in the buffer solution, each protein having its own optimum. The 57 kDa protein was indistinguishable by size, binding properties and immunoprecipitation from the polypyrimidine tract binding protein (PTB), first described as a nuclear protein binding to the polypyrimidine tracts (PPTs) in introns. Cross-competition experiments showed that leader 3 has a much higher affinity for this 57 kDa protein than the PPT on which PTB was originally characterized. By competition with different fragments of leader 3, we were able to localize the binding of the 57 kDa protein to a 162 nt RNA fragment (AsnI-PvuII) in the 3'-part of the leader. When placed before a chloramphenicol acetyltransferase (CAT) open reading frame, this RNA fragment stimulated translation in reticulocyte lysate 3-fold, while other fragments of leader 3 repressed translation. The efficient translation directed by the 162 nt AsnI-PvuII fragment fused to CAT could be repressed by adding free AsnI-PvuII RNA fragment, indicating that the high translation efficiency of the AsnI-PvuII-CAT synthetic mRNA was due to the binding of protein and not to the structure of the RNA itself.
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PMID:Proteins binding to the leader of the 6.0 kb mRNA of human insulin-like growth factor 2 influence translation. 771 79

Transcriptional regulation of the interleukin-5 (IL-5) gene in T lymphocytes appears to be of central importance in the control of the eosinophilia characteristic of allergic responses and certain parasite infections. Previous studies of IL-5 gene regulation have been hampered by the lack of a transfection assay, which detects the antigen-responsive enhancer in the IL-5 promoter. Here we show that stable transfection of the Th2 clone D10.G4.1 and the T lymphoma EL4.23 with chloramphenicol acetyltransferase reporter gene constructs carrying the region to -3859 gives inducible expression with the known regulatory characteristics of the endogenous IL-5 gene. To facilitate detailed analysis of the promoter region, 3.9 kb of DNA sequence immediately up stream of the start of transcription was determined and the minimum upstream region required for inducible expression was further localized, by stable transfection studies in EL4.23 cells, to the region up to -1016. A CTF/NF1 site in the upstream enhancer at -940 to -928 was shown to be required for regulated inducible expression. Mutation of this sequence motif abolished inducibility and also prevented binding of the sequence to a nuclear protein(s). A TCATTT-containing element in the proximal promoter region was also demonstrated to be essential for inducible expression of the IL-5 gene, similar to the role of this conserved element in the transcriptional regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 genes.
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PMID:Localization of the inducible enhancer in the mouse interleukin-5 gene that is responsive to T-cell receptor stimulation. 771 77


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