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 level of fibronectin (FN) gene expression is very high in resting rat 3Y1 cells but greatly decreased in adenovirus E1-transformed cells. To study the mechanism of this down-regulation, nuclear factors binding to the 5'-flanking region of the FN gene were analyzed by gel retardation assay and DNase I footprinting. Nuclear factors that were present in the transformed cells but nearly absent in resting 3Y1 cells interacted with multiple sites of the promoter region. Oligonucleotide competition with the FN promoter-chloramphenicol acetyltransferase (CAT) reporter constructs (pFCAT) for these factors in the transformed cells indicated that all of them had a negative effect on FN gene expression. Of them, a factor(s) (G10BP) binding to the G10 stretch from positions -239 to -230 and to two GC boxes consisting of the G10 stretch with one internal C residue insertion from positions -105 to -95 and -54 to -44 had the strongest repressive activity. Introduction of substitutive mutations into these G-rich sequences resulted in the increase in CAT activity of pFCAT in the transformed cells. The recognition sequences of G10BP and Sp1 overlap in two GC boxes. G10BP has stronger affinity for heparin and GC boxes than does Sp1, suggesting that G10BP may repress FN gene transcription by displacing Sp1.
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PMID:Induction of E1A-responsive negative factors for transcription of the fibronectin gene in adenovirus E1-transformed rat cells. 140 98

When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process. To investigate early events in adipocyte development, we have focused on the transcriptional activation of the LPL gene. For this purpose, we have cloned and fused different parts of intragenic and flanking sequences with a chloramphenicol acetyltransferase reporter gene. Transient transfection experiments and DNase I hypersensitivity assays indicate that several positive as well as negative elements contribute to transcriptional regulation of the LPL gene. When reporter gene constructs were stably introduced into preadipocytes, we were able to monitor and compare the activation patterns of different promoter deletion mutants at selected time points representing the process of adipocyte development. We could delimit two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro. These elements, LP-alpha (-702 to -666) and LP-beta (-468 to -430), contain a striking similarity to a consensus sequence known to bind the transcription factors HNF-3 and fork head. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicate that factors with DNA-binding properties similar to those of the HNF-3/fork head family of transcription factors are present in adipocytes and interact with LP-alpha and LP-beta. We also demonstrate that LP-alpha and LP-beta were both capable of conferring a differentiation-linked expression pattern to a heterolog promoter, thus mimicking the expression of the endogenous LPL gene during adipocyte differentiation. These findings indicate that interactions with LP-alpha and LP-beta could be a part of a differentiation switch governing induction of the LPL gene during adipocyte differentiation.
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PMID:Characterization of the human lipoprotein lipase (LPL) promoter: evidence of two cis-regulatory regions, LP-alpha and LP-beta, of importance for the differentiation-linked induction of the LPL gene during adipogenesis. 140 52

The plasma cholesteryl ester transfer protein (CETP), primarily synthesized in the liver of several species, is expressed at very low levels in a number of transformed human liver cell lines. The human CETP gene promoter contains a sequence that closely resembles the binding site for the transcription factor CCAAT/enhancer-binding protein (C/EBP). This site is capable of binding C/EBP, as shown by electrophoretic mobility shift and DNase I footprint analyses. Transient expression of the bacterial chloramphenicol acetyltransferase gene under the control of the human CETP gene promotor gave low activities in the human hepatoma cell line, HepG2. However, in the presence of C/EBP, CAT activity was markedly elevated indicating that CETP gene promoter activity was enhanced. In primary cultures of isolated hepatocytes, CETP mRNA was lost rapidly and in parallel with the C/EBP mRNA. C/EBP may play an important role in the proper maintenance of CETP gene promoter activity, and its low levels in proliferating or cultured cells may account for the low level of the CETP gene expression in immortalized human liver cell lines or cultured hepatocytes.
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PMID:The CCAAT/enhancer-binding protein trans-activates the human cholesteryl ester transfer protein gene promoter. 142 86

We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression. Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis.
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PMID:A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. 144 77

The 5'-flanking region of the human lactoferrin gene was isolated from a human placental genomic library. This genomic clone contains a 16-kilobase pair (kbp) insert and produces seven fragments when digested with the SacI restriction enzyme. We sequenced one of the fragments that comprises 1294 bp of the 5'-flanking sequence, 79 bp of the first exon, and 690 bp of the first intron. A major transcription start site was mapped by primer extension. The region immediately upstream from the transcription initiation site following the first exon is abundant in G and C nucleotides. In the promoter and 5'-flanking region within a 300-bp stretch (-465 to -165) of the DNA, we found a noncanonical TATA box (ATAAA), CAAT-like sequence (CAAC) and sequences homologous to the consensus SP1 binding site, Pu.1/Sp.1 binding element (PU box), two half-palindromic estrogen response elements (EREs; GGTCA), an imperfect ERE (GGTCAAGGCGATC), and a sequence resembling the chicken ovalbumin upstream promoter transcription factor (COUP-TF) binding site (GTCTCACAGGTCA). The COUP-TF binding site and the imperfect ERE shared five nucleotides (GGTCA). With the exception of the two half-palindromic EREs, the elements with very well matched sequences were also found in the corresponding positions in the mouse lactoferrin gene. The synthetic oligonucleotide, including the 26 bp of COUP/ERE sequence, was cloned before the SV40 promoter in a chloramphenicol acetyltransferase reporter construct. These chimeric plasmids were transiently transfected into human endometrium carcinoma RL95-2 cells to assess hormone responsiveness. We found that the COUP/ERE element acted as an enhancer in response to estrogen stimulation. In vitro DNase I footprinting analysis showed binding of the estrogen receptor on the imperfect ERE. In contrast to the mouse lactoferrin COUP/ERE element, COUP-TF does not interact with this element, as demonstrated by band shift assay and site-directed mutagenesis. Therefore, the molecular mechanisms of the estrogen action that govern the lactoferrin gene expression differ between mouse and human.
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PMID:Differential molecular mechanism of the estrogen action that regulates lactoferrin gene in human and mouse. 148 Jan 83

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

We cloned, sequenced and characterized a promoter region of the mouse homologue of the Alzheimer's disease amyloid precursor protein (APP)-encoding gene. The promoter region is highly homologous to that of the human APP (hAPP) gene. It has a high G+C content, lacks typical 'TATA' and 'CAAT' boxes, and contains possible binding sites for AP-1, heat-shock factor, Sp1 and AP-4. The promoter region was fused with the cat reporter gene, and the fusion genes were transfected to both the NB41A3 (mouse neuroblastoma) and L-cell lines. The promoter activity was monitored by chloramphenicol acetyltransferase (CAT) activity in a transient expression assay. The promoter was equally active in both cell lines. The deletion analysis revealed that there existed a negative regulatory element(s) between -153 and -100 bp and a positive element(s) between -100 and -37 bp. The negative element was shown to suppress the transcriptional activity of heterologous simian virus 40 promoter. DNase I footprinting experiments revealed that three nuclear protein-binding sites existed in the regulatory region, one in the negative and two in the positive regulatory regions. Gel retardation assay showed that Sp1 was one of the factors binding to the positive regulatory region. A nuclear factor binding to the negative regulatory region seemed to be missing in brain.
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PMID:Positive and negative regulatory elements for the expression of the Alzheimer's disease amyloid precursor-encoding gene in mouse. 155 68

To further define the transcriptional regulation of the P38 promoter in the minute virus of mice (MVM) genome, we constructed a series of internal deletion and linker scanning mutations. The mutant P38 constructs were assayed for transcriptional activity in vitro by primer extension analysis with nuclear extracts from murine A92L fibroblasts. Mutations which disrupted the GC box and TATA box severely reduced transcription in vitro. DNase I footprinting analysis confirmed that the murine transcription factor Sp1 bound to the GC box; however, no factors were observed interacting with a putative transcriptional activation regulatory element, termed the TAR element. The linker scanning mutations were analyzed in vivo by using a chloramphenicol acetyltransferase expression assay system, in both the presence and absence of constructs expressing the viral nonstructural protein, NS1. The ability of NS1 to transactivate the P38 promoter (up to 1,000-fold) depended entirely on the presence of intact GC and TATA box sequences. Disruption of the TAR element by either linker insertion mutations or an internal deletion did not inhibit transactivation of the P38 promoter. These results suggest that NS1 transactivates the P38 promoter indirectly by interacting with one or more components of the P38 core-transcription complex.
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PMID:The GC box and TATA transcription control elements in the P38 promoter of the minute virus of mice are necessary and sufficient for transactivation by the nonstructural protein NS1. 158 30

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that inhibits both tissue-type and urokinase-type plasminogen activators. Expression of PAI-1 is regulated by growth factors, cytokines, and hormones. To determine the molecular mechanisms involved in the basal expression of the rat PAI-1 gene, we have analyzed the cis-acting sequences and the trans-acting factors involved in the transcription of this gene in the HTC rat hepatoma cell line. DNase I protection analyses revealed eight regions within the first 764 base pairs of 5'-flanking sequence that interact specifically with HTC cell nuclear proteins. The proteins that bind to five of the eight footprinted sites were identified as PEA3-, Sp1-, and CTF/NF-1-like proteins using competition electrophoretic mobility shift assays. The expression of fusion genes containing progressive 5' deletions of the rat PAI-1 promoter linked to the chloramphenicol acetyltransferase reporter gene were analyzed in transient transfection experiments in HTC cells. These studies demonstrated the Sp1 and CTF/NF-1 sites to be important for transcriptional activation. Two of the footprinted sites contain the sequence 5'-TTTGn(n)TCAAT-3' and were shown in competition electrophoretic mobility shift assays to bind the same or related protein(s). Sequences containing these sites, from -764 to -628 base pairs, and from -266 to -188 base pairs, were identified in functional studies as repressor elements of transcription.
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PMID:Regulatory sequences and protein-binding sites involved in the expression of the rat plasminogen activator inhibitor-1 gene. 160 87

To evaluate the function of the murine ornithine decarboxylase (ODC) gene promoter, expression of chimeric ODC-chloramphenicol acetyltransferase (CAT) plasmids (pODCcat) containing 1,658 nt of the ODC promoter sequence and its various 5'-deletions was analyzed. In transient expression assays with NIH/3T3 mouse cells, pODCcat constructs exhibited fairly strong promoter activity yielding CAT values up to 40% of those obtained with the viral promoter RSV. Interestingly, 5'-deletions of the pODCcat constructs increased the promoter activity over that achieved using the entire 1.6-kb 5'-flanking region, with the highest activity being observed with about 750 nt of the ODC promoter. This finding suggests that the distal part of the promoter includes DNA elements which are involved in repressing its function. The promoter region could be deleted down to the proximal 97 nt and still be stimulated by cAMP to the same extent as the 1.6-kb promoter. DNase I footprinting and methylation interference studies showed that a specific protein binds to the region from -59 to -39, which encompasses a DNA motif resembling the consensus cyclic AMP response element (CRE). However, comparative gel retardation and Southwestern blotting experiments with the putative ODC-CRE and the somatostatin promoter CRE indicated that the 70-kDa protein interacting with the CRE-like element of the ODC promoter is different from the well-characterized nuclear CRE-binding protein CREB.
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PMID:Protein-DNA interactions in the cAMP responsive promoter region of the murine ornithine decarboxylase gene. 165 Apr 55


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