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)

Nuclear protein binding to the human interferon-gamma (IFN-gamma) promoter was investigated to determine the structural basis for the control of gene expression during T cell activation. DNase I footprinting of gel-shift complexes demonstrated that proteins bind to two downstream (-124 to -114 and -36 to -30) and one upstream (-534 to -486) element in the IFN-gamma gene promoter. Treatment of human peripheral blood lymphocytes or continuous T cell tumors with phorbol 12-myristate 13-acetate (PMA) plus phytohemagglutinin or calcium ionophore results in a pattern of response that is similar when using either the upstream or downstream elements. Upon induction of T cells, the lower mobility gel-shift band disappears. Yet the equivalent band which is also present in non-T cells is unperturbed after PMA + calcium ionophore treatment. The higher mobility band which is modified upon induction is restricted to the T cell lineage. Upstream and downstream elements share similar protein-binding motifs as indicated by the homology of footprinted sequences, the similarity of protein-binding patterns, and the ability of these elements to compete against each other in gel-shift protein-binding assays. Protein binding to the downstream elements appears to be interactive, since both sites are required for complex formation. When either of the two downstream elements is disrupted by site-directed mutagenesis, the higher mobility gel-shift band is diminished by an amount that is consistent with the reduction in reporter (chloramphenicol acetyltransferase) gene expression. Therefore, proteins in the ubiquitous gel-shift band appear to be associated with the inactive state of IFN-gamma, while the modified band is closely associated with the positive regulation of IFN-gamma gene expression.
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PMID:Characterization of nuclear protein binding to the interferon-gamma promoter in quiescent and activated human T cells. 151 29

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EmukCAT, contains the Ig heavy chain enhancer (Emu) and the kappa light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, delta EmukCAT, CAT is under the control of the kappa promoter alone. Emu and kappa relative activity were assessed by CAT assay. In EmukCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In delta EmukCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon-gamma (IFN-gamma) increased CAT expression to varying extents in cells derived from EmukCAT mice but not in spleen cells prepared from delta EmukCAT mice. Thus, the presence of Emu, in addition to the kappa promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EmukCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN-gamma caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.
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PMID:Activation of immunoglobulin control elements in transgenic mice. 172 70

Regulation of human type I procollagen gene expression was studied in cultured fibroblasts both at the transcriptional and posttranscriptional level. Transcriptional regulation was examined in cultures transfected with a human pro alpha 2(I) collagen promoter/reporter gene (chloramphenicol acetyltransferase) construct, while posttranscriptional regulation was assessed by parallel determinations of type I procollagen mRNA steady-state levels. Transforming growth factor-beta 1 (TGF-beta 1) elicited a marked, approximately 5-23-fold, enhancement of pro alpha 2(I) collagen promoter activity, which was accompanied by an elevation of type I procollagen mRNA levels. This enhancement of gene expression was suppressed by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), as determined at mRNA steady-state level, but two distinct mechanisms were involved. TNF-alpha suppressed the pro alpha 2(I) collagen promoter activity, whereas IFN-gamma had only a minimal effect at transcriptional level. The effects of TNF-alpha and IFN-gamma were synergistic, suggesting that combination of these two factors may potentially provide pharmacologic means to counteract tissue deposition of collagen in diseases involving TGF-beta.
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PMID:Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels. 212 79

In this report, we determined that induction of the DR alpha-chain by recombinant human interferon-gamma (IFN-gamma) in a human glioblastoma multiform cell line is transcriptionally regulated and showed that protein synthesis is not necessary for this to occur. The regions of the DR alpha-chain gene that are responsible for basal and recombinant IFN-gamma-induced gene transcription have been determined by gene transfer of a series of 5' deletion mutants in which the upstream region of the DR alpha chain was linked to a reporter gene, chloramphenicol acetyltransferase. Chloramphenicol acetyltransferase transcript and protein levels were determined by S1 nuclease protection and chloramphenicol acetyltransferase enzyme assays, respectively. By using these deletion mutants, we were able to draw the following conclusions. (i) One hundred and nine base pairs of upstream sequence contains the basic DR alpha-chain gene promoter and represents the minimal amount of sequence necessary for basal gene expression. (ii) An additional 9 base pairs of upstream sequence can mediate recombinant IFN-gamma induction. (iii) Maximal recombinant IFN-gamma induction requires at most an additional 23 base pairs of upstream sequence. (iv) The sequence between positions -267 and -141 does not appear to contain any additional positive or negative regulatory elements. These results suggest that the region between positions -141 and -109 contains a critical IFN-gamma-responsive element. Substitution mutagenesis was performed to confirm this suggestion.
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PMID:Detailed delineation of an interferon-gamma-responsive element important in human HLA-DRA gene expression in a glioblastoma multiform line. 284 68

The expression of class II major histocompatibility (MHC) antigens is central to the mounting of a successful immune response. Understanding the molecular mechanisms involved in the induction of class II MHC expression may therefore provide the knowledge necessary to manipulate the immune system appropriately. We are particularly interested in the induction of class II MHC antigens on cells in the brain, because of the potential involvement of such brain cells in the initiation and perpetuation of autoimmune-like diseases of the central nervous system. We examined the mechanisms involved in interferon-gamma (IFN-gamma) induction of class II MHC antigens on a human glioblastoma multiforme cell line. This paper describes the identification of a 297-bp (base pair) fragment of the class II MHC DR alpha chain gene which is involved in IFN-gamma induction. We were able to identify this IFN-gamma responsive sequence by preparing recombinant plasmids containing 5' flanking pieces of the human DR alpha chain gene placed upstream of the indicator gene, chloramphenicol acetyltransferase (CAT). These recombinant plasmids were transfected into human glioma cells which were then cultured in the presence or absence of IFN-gamma. After 48 hr, transient expression of CAT was assayed by thin layer chromatography. CAT enzyme activity was significantly increased only in IFN-gamma-treated cells. This increase was also reflected at the RNA level in that IFN-gamma treatment resulted in higher CAT transcripts. A computer homology search revealed a possible consensus sequence shared among different IFN-gamma-inducible genes.
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PMID:Identification of an interferon-gamma response region 5' of the human histocompatibility leukocyte antigen DR alpha chain gene which is active in human glioblastoma multiforme lines. 302 77

In the present report we describe a heretofore unrecognized role for a Jak/STAT signaling pathway, namely the stimulation of expression of the aromatase P450 (CYP19) gene, and hence of estrogen biosynthesis, in human adipose tissue. Expression of this gene in adipose tissue as well as in adipose stromal cells maintained in the presence of serum and glucocorticoids is regulated by a distal TATA-less promoter, I.4, which contains a glucocorticoid response element, an Sp1 binding site, and an interferon-gamma activation site (GAS) element. The stimulatory action of serum (in the presence of dexamethasone) can be replaced by interleukin (IL)-11, leukemia inhibitory factor, and oncostatin-M, as well as by IL-6, providing the IL-6 soluble receptor is also present. Stimulation of the cells by these factors led to rapid phosphorylation of Jak1, but not Jak2 or Jak3, on tyrosine residues. STAT3 but not STAT1 was also phosphorylated and bound to the GAS element in the I.4 promoter region. When regions of this promoter were fused upstream of the chloramphenicol acetyltransferase reporter gene and transfected into the cells, mutagenesis or deletion of the GAS element led to complete loss of reporter gene expression. Since adipose tissue is the major site of estrogen biosynthesis in men and in postmenopausal women, this pathway involving a Jak/STAT signaling mechanism acting together with glucocorticoids and Sp1 appears to be the principal means whereby estrogen biosynthesis is regulated in the elderly.
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PMID:Aromatase P450 gene expression in human adipose tissue. Role of a Jak/STAT pathway in regulation of the adipose-specific promoter. 760 17

Growth hormone activates gene transcription of the serine protease inhibitors (SPI) 2.1 and 2.2 by an unknown mechanism. In order to define the promoter regions responsible for this effect and to characterize the transcription factors involved, we have performed gel electrophoresis mobility shift assays on nuclear extracts from cell lines transfected with growth hormone receptor cDNA. We have identified a 9-base pair DNA element, the SPI-GLE 1, which forms a complex with nuclear proteins following activation by growth hormone and which, when placed upstream of a minimal thymidine kinase promoter, drives chloramphenicol acetyltransferase expression in a growth hormone-dependent fashion. This element is similar to those from several genes regulated by other cytokines including interferon. The growth hormone-induced complexes formed were dependent on tyrosine phosphorylation but did not contain the interferon-gamma-activated transcription factor Stat 91. Competition studies with oligonucleotides similar to the SPI-GLE 1 reveal the sequence of a consensus element that specifically binds growth hormone-regulated nuclear proteins.
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PMID:Growth hormone specifically regulates serine protease inhibitor gene transcription via gamma-activated sequence-like DNA elements. 792 35

The fluoroquinolone antibiotic, ciprofloxacin (cipro), induces hyperproduction of interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) in stimulated human peripheral blood lymphocytes. In this investigation an enhanced and prolonged IL-2 and IL-2 mRNA response was also detected in both stimulated (T cell mitogens or alloantigens) murine splenocytes and in the stimulated murine T cell line EL-4 in the presence of ciprofloxacin (5-80 micrograms/ml) as compared to control cells without antibiotics. However, in contrast to human lymphocytes, IFN-gamma production was inhibited and IFN-gamma mRNA levels were unaffected at 24 h and only slightly upregulated at 48 and 72 h of culture in murine splenocytes incubated with cipro (20 micrograms/ml). EL-4 cells were transfected with a plasmid containing the IL-2 promoter and enhancer region linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Analysis of CAT activity revealed that cipro enhanced IL-2 gene induction. In addition, EL-4 cells incubated with ciprofloxacin showed an early peak and more activated nuclear factor of activated T cells (NFAT-1) as compared to control cells without antibiotics. Cipro did not affect the nuclear transcription factors AP-1 or NFIL-2A. Taken together, cipro inhibited IFN-gamma synthesis, but enhanced IL-2 production in murine lymphocytes by means of influencing NFAT-1 and causing an increased IL-2 transcription.
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PMID:Increased interleukin 2 transcription in murine lymphocytes by ciprofloxacin. 801 29

The ribosome binding site (RBS) of prokaryotic mRNA is divided into 5' and 3' portions by the translation initiation codon. Although it is well known that the presence of an appropriate RBS containing only the 5' portion is sufficient to direct the initiation of protein synthesis, the 3' portion appears to play a significant role in modulating the initiation process as well. Here we examine the influence of adenine-rich motifs frequently found in the 3' portion of highly expressed prokaryotic mRNAs. Two synthetic DNA fragments, GAGAAAAAAATC (corresponding to the first 12 nucleotides following the initiation codon of the chloramphenicol acetyltransferase gene), and AAAAAAATTAA were used to modify the beginning of the coding region of the human immune interferon-gamma (IFN-gamma) gene. The level of the protein synthesis in Escherichia coli directed by plasmids containing these constructs was quantitated. We found that placing either adenine-rich motif in the 3' portion of the RBS strongly enhanced gene expression, probably through an effect on translation initiation. We have also compared the protein expression levels of these gene constructs containing different series of 5'-RBSs with varying precistronic lengths and Shine-Dalgarno sequence lengths. The results suggest a positive functional role for the 3' adenine-rich motif. A possible mechanism for these effects is discussed.
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PMID:The influence of adenine-rich motifs in the 3' portion of the ribosome binding site on human IFN-gamma gene expression in Escherichia coli. 802 37

Type II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) are capable of decreasing the levels of alpha 1(II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-gamma on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-gamma decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of alpha 1(II) procollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5'-flanking sequences of COL2A1 (-577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-gamma. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-gamma is mediated primarily at the transcriptional level.
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PMID:Transcriptional suppression by interleukin-1 and interferon-gamma of type II collagen gene expression in human chondrocytes. 812 89


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