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)

In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.
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PMID:Promoter region of the human alpha 2A adrenergic receptor gene. 138 31

The promoter regions of the three mammalian transforming growth factor-beta genes (TGF-beta s 1, 2, and 3) have been recently cloned and characterized. The sequences show little similarity, suggesting different mechanisms of transcriptional control of these genes. To study differences in transcriptional regulation of mammalian and avian TGF-beta, we have cloned and sequenced the 5'-flanking region of chicken TGF-beta 3. Characterization of this region showed a TATA box and cAMP-responsive element (CRE) and AP-2 binding site consensus sequences starting at 12 and 28 base pairs, respectively, upstream from the TATA box. Moreover, four additional AP-2-like sites, 10 binding sites for the transcription factor Sp1, as well as two AP-1-like sites were also identified. Except for 32 base pairs of identity centered around the TATA box and CRE site and four other relatively small regions of identity, the chicken TGF-beta 3 promoter was found to be structurally very different from the human TGF-beta 3 promoter. Promoter fragments were cloned into a chloramphenicol acetyltransferase reporter plasmid to study functional activity. Basal transcriptional activity of the promoter was regulated in quail fibrosarcoma QM7 cells and in human adenocarcinoma A375 cells by multiple upstream elements including the TATA, CRE, and AP-2 sites. As in the human TGF-beta 3 promoter, the CRE site showed activation by forskolin, an effect which could be shown by expression of TGF-beta 3 mRNA in cultured chicken and quail cells as well. Our results indicate a complex pattern of transcriptional regulation of the chicken TGF-beta 3 gene and suggest that differences in the regulation of expression of the genes for mammalian and avian TGF-beta 3 may result in part from the unique structure of their 5'-flanking regions.
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PMID:Identification and characterization of the chicken transforming growth factor-beta 3 promoter. 140 6

SP-A is an abundant pulmonary surfactant-associated protein whose expression is controlled in a cell- and developmental-specific manner. To analyze regulation of SP-A gene expression, the murine SP-A gene was cloned and sequenced. The murine DBA/2J gene was approximately 4.6 kb in length comprised of six exons and five introns. Three mRNAs of 3.0, 1.7, and 0.9 kb were detected by Northern blot analysis of murine lung mRNA. Expression of the SP-A mRNAs was first detected at day 15 of gestation and increased dramatically before birth. A single SP-A gene was detected in the DBA/2J mouse genome. SP-A mRNA was detected in lung but not in the gastrointestinal tract, kidney, brain, liver, or heart and was detected by in situ hybridization in bronchial and alveolar cells of the murine lung. Primer extension analysis with a primer to exon three revealed two extension products differing by 9 bp in length, suggesting two closely juxtaposed transcription initiation sites. Chimeric gene(s) containing 1.8 kb of 5' SP-A sequences and the bacterial chloramphenicol acetyltransferase gene were expressed in pulmonary adenocarcinoma cells and in HeLa cells. Expression of the murine SP-A gene is partially controlled by non-cell-selective transcriptionally active sequences.
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PMID:Murine pulmonary surfactant SP-A gene: cloning, sequence, and transcriptional activity. 144 58

The human prothrombin gene is expressed predominantly in hepatocytes. Previous work indicated that this tissue specificity is transcriptionally regulated. In order to identify the cis-acting regulatory elements in the 5' flanking region of the human prothrombin gene which may direct the expression of prothrombin in hepatocytes, a series of hybrid plasmids were constructed linking portions of the 5' flanking region of the human prothrombin gene to the bacterial chloramphenicol acetyltransferase gene. Expression of these hybrid plasmids was examined in calcium phosphate-mediated transient transfections of HepG2 cells, a human hepatoblastoma cell line which expresses prothrombin, and HeLa cells, an adenocarcinoma cell line which does not express detectable amounts of prothrombin. Both the prothrombin promoter and an upstream regulatory region containing sequence homologous to the hepatocyte nuclear factor 1 (HNF-1) binding site (nucleotides -919 to -790 relative to the prothrombin transcription initiation site) were required for expression in HepG2 cells. The upstream region also exhibited non-tissue-specific enhancer activity. Gel mobility shift assays confirmed cell-type-specific differences in the protein-DNA interactions between proteins in HepG2 or HeLa nuclear extracts and either the promoter region or the upstream regulatory region of the gene.
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PMID:The human prothrombin gene: transcriptional regulation in HepG2 cells. 146 33

The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5'-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5'-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.
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PMID:Isolation and characterization of the human sucrase-isomaltase gene and demonstration of intestine-specific transcriptional elements. 156 17

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.
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PMID:cis-acting elements that confer lung epithelial cell expression of the CC10 gene. 163 15

Transforming growth factor beta (TGF-beta) isoforms inhibit the growth of many cell types and block progression of the cell cycle by inhibiting events in late G1 phase. The retinoblastoma gene product, RB, also has properties of a cell-cycle regulatory factor. It remains underphosphorylated in the presence of TGF-beta and has been shown to repress the activity of the c-fos promoter, resulting in inhibition of transit through the cell cycle. These observations led us to examine effects of human RB on the expression of the human TGF-beta 1 gene. Using chimeric TGF-beta 1 promoter-chloramphenicol acetyltransferase gene constructs, we show that RB induces TGF-beta 1 gene expression in CCL-64 mink lung epithelial cells and A-549 human lung adenocarcinoma cells but represses its expression in NIH 3T3 and AKR-2B mouse cells. Several sequences homologous to the c-fos RB control element were identified in the TGF-beta 1 promoter. These results demonstrate that human RB can regulate TGF-beta 1 gene expression negatively or positively depending on the cell type.
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PMID:Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene. 190 52

Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.
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PMID:Expression of recombinant androgen receptor in cultured mammalian cells. 217 2

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.
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PMID:Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells. 749 77

Pulmonary surfactant protein B (SP-B) is required for normal surfactant function and for survival at birth. To further study SP-B gene expression, we sequenced genomic clones and examined promoter activity of SP-B DNA fragments by transient transfection. A plasmid construct containing human SP-B fragment -1039/+431 linked to chloramphenicol acetyltransferase (CAT) reporter gene was readily expressed in H441 cells, which are derived from a human lung adenocarcinoma, but was < 4% as active in Hep G2, HeLa, and Calu 6 cell lines. SP-B promoter activity in H441 cells was orientation dependent and increased by linked Rous sarcoma virus (RSV) enhancer and was stronger than for thymidine kinase (tk) and RSV promoters. Deletional mapping of the 5' flanking region with exonuclease III suggested nonspecific negative (-811/-1039)- and positive (-453/-641)-control regions and a cell-specific enhancer region at -208 to -54. When a fragment from -403 to -35 base pairs (bp) was placed upstream or downstream of tkCAT, in either orientation, expression in H441 cells but not other cell lines was increased 4- to 28-fold relative to tkCAT. Deletional analysis of the 3' terminus indicated a requirement for at least 7 bp 3' of the transcription start site. Promoter activity was strongly inhibited in a dose-dependent fashion by phorbol ester, with responsiveness mapped to bp -208/-54, but was not responsive to glucocorticoid.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the promoter of human pulmonary surfactant protein B gene. 773 8


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