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)

A plasmid carrying the 5'-flanking region (-1584 to +47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5- to 9-fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of E1A resides in a common region(s) of 13S and 12S E1A products. The major target region of E1A was mapped within the 68 base-pair region (-21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2F, although the upstream region (-83 to -21) including ATF(CREB)-binding consensus had an additional effect in the transactivation.
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PMID:Activation of the mouse proliferating cell nuclear antigen gene promoter by adenovirus type 12 E1A proteins. 135 54

A plasmid carrying the 5'-flanking region (-1852 to +33 with respect to the transcription initiation site) of the mouse DNA polymerase beta gene fused with the chloramphenicol acetyltransferase (CAT) gene was cotransfected into mouse N18TG2 cells with adenovirus type 12 E1 genes-expressing plasmids. Expression of E1A gene products resulted in the elevation of the CAT expression by 3 to 7 folds, but that of E1B gene product was much less effective. RNase protection analysis revealed that the activation by E1A was at the transcription process. Both the 13S E1A and the 12S E1A activated the DNA polymerase beta gene promoter, indicating that the activation domain of E1A is in a common region(s) of 13S and 12S E1A products. The major target sequence of E1A was mapped within the 10 base pair-region (-30 to -20) of the DNA polymerase beta gene promoter, which overlapped with the palindromic sequence known as the ATF(CREB)/E4F-binding consensus. The results suggest that the palindromic sequence is essential for E1A-induced transcriptional activation of the mouse DNA polymerase beta gene.
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PMID:Activation of the mouse DNA polymerase beta gene promoter by adenovirus type 12 E1A proteins. 153 5

The expression of the human beta interferon (IFN-beta) gene was activated by the adenovirus E1B-19K protein. The sequence within the IFN-beta promoter which is related to the activation was analyzed by chloramphenicol acetyltransferase (CAT) assay. The repeated hexamer units, present within the region between -109 and -65 relative to the cap site, were required for the activation of the IFN-beta gene by the E1B-19K protein. The (AAGTGA)8 region, as a typical hexamer of the consensus sequences, was tested for function in the activation by the E1B-19K protein. When the hexamer (AAGTGA)4-8 was inserted upstream of several reporter genes (such as p55cat, pdlE1A-CAT, and pE1B-CAT) which were inefficiently stimulated, the CAT activities of these fusion genes were efficiently stimulated by the E1B-19K protein. These results show that the tandemly repeated hexamer sequences within the IFN-beta promoter can function as an inducible regulatory element in the activation by the adenovirus E1B 19K protein.
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PMID:Tandemly repeated hexamer sequences within the beta interferon promoter can function as an inducible regulatory element in activation by the adenovirus E1B 19-kilodalton protein. 213 95

In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.
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PMID:Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation. 247 19

The plasmid construct pSVO-CAT has been used to test adenovirus promoter activities in the unmethylated or methylated state. We have now observed that the E2A late promoter of adenovirus type 2 (Ad2) DNA also activated the chloramphenicol acetyltransferase (CAT) gene upon transfection of the pAd2E2A-CAT construct into mammalian cells, and it was inactivated by specific methylations of three 5' -CCGG- 3' sites. Similar results had been reported previously after microinjecting promoter-methylated constructs into oocytes of Xenopus laevis. Surprisingly, it was found that the pSVO-CAT construct, which lacked eukaryotic promoter sequences, was able to express the CAT gene upon transfection into human or hamster cells that harbored and constitutively expressed the E1 region of Ad2 or Ad5 DNA. In these cells, the expression of the pAd2E2A-CAT construct was enhanced, but it was only partly sensitive to DNA methylation, possibly because DNA methylation was counteracted directly or indirectly by E1 functions. The pSVO-CAT construct was also expressed in HeLa or BHK21 cells upon cotransfection with a plasmid carrying the HindIII-G fragment of Ad2 DNA that contained the E1A region and part of the E1B region. By mapping pSVO-CAT-specific RNAs, we could demonstrate that pSVO-CAT activity in Ad2- or Ad5-transformed cells was mediated by prokaryotic promoter-like sequences in the pBR322 section of the construct, and it presumably functioned via trans-activation mediated by the E1 region. This trans-activation of pSVO-CAT in adenovirus-transformed cells was partly insensitive to DNA methylation.
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PMID:Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5' -CCGG- 3' methylation. 293 60

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in insect cell lines in culture. In mammalian cells, however, the virus cannot be propagated. AcNPV DNA does not replicate or persist and is not transcribed in mammalian cells (Tjia et al., 1983). In insect cells productively infected with AcNPV, at least two major late viral gene products have been recognized, the polyhedrin, which makes up the bulk of the polyhedral inclusion bodies in infected cell nuclei, and a 10,000 Da protein (p10) of unknown function. The p10 promoter has been fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as a reporter gene (Knebel et al., 1985). Activity of this construct can be elicited in AcNPV-infected insect cells but not in uninfected insect cells or in mammalian cells. Presumably, the late p10 promoter requires other AcNPV gene products for activity. When the pAcp10-CAT construct is transfected into BHK21 hamster cells at about 18 h after infection with human adenovirus type 5 (Ad5), the insect AcNPV promoter is transactivated in cells of the heterologous mammalian species. The results of S1 protection analyses on the RNA from Ad5-infected and pAcp10-CAT transfected cells reveal that the p10 promoter is used for initiation of transcription. Similarly, the p10 insect virus promoter is activated in BHK21 hamster cells cotransfected with the HindIII-G fragment of adenovirus type 2 (Ad2) DNA which contains the E1A and parts of the E1B region in the left terminal 7.8% of the Ad2 genome. Moreover, in human 293 cells or in BHK297-C131 hamster cells, which both carry and constitutively express the E1 region of Ad5 DNA, the pAcp10-CAT construct is also expressed, and similarly in HE7 hamster cells which carry appreciable portions of the Ad2 genome (Klimkait and Doerfler, 1985). It is concluded that adenovirus functions are capable of transactivating a heterologous insect virus promoter in mammalian cells.
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PMID:Activation of an insect baculovirus promoter in mammalian cells by adenovirus functions. 296 53

The left-most 3.9 kb of adenovirus type 40 (Ad40) DNA has been sequenced using cloned viral DNA fragments. The Ad40 E1 region is deduced to code for at least four polypeptides, 221 and 249 amino acids as E1A products in addition to 166 and 475 amino acids as E1B products. E1B polypeptides share about 50% homology with well-defined adenovirus types, 2/5, 7, and 12, throughout the E1B sequences. E1A homology of Ad40 to these types is relatively lower than that of E1B, while highly conserved regions of E1A are retained to a certain level in Ad40 as well. Activity for morphological transformation of Ad40 E1A on 3Y1 cells is considerably lower when compared to that of Ad5 and Ad12 E1A genes. Transient chloramphenicol acetyltransferase (CAT) expression assay shows that Ad40 E1A has a trans-acting function, though lower than that of other E1A genes, on adenovirus early promoter. The Ad40 E1A promoter also holds only a little cis-acting activity in 3Y1 cells. Lower activities of both Ad40 E1A promoter and certain E1A functions may explain in part the difficulty in propagation of Ad40.
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PMID:Characterization of adenovirus type 40 E1 region. 296 14

As an alternative to directing plant or bacterial toxins to surface receptors, we are investigating the possibility of killing tumor cells by the expression of an exogenously introduced toxin gene (i.e., cell suicide). Tissue-specific gene regulatory elements might thus be exploited to achieve selective killing. To assess the feasibility of such an approach, we have transfected human cells (HeLa, B-lymphoblastoid, and 293 cells) with plasmids containing the diphtheria toxin A-chain (DT-A) coding sequence. The presence of the DT-A sequence lowered the level of transient expression of chloramphenicol acetyltransferase from a cotransfected plasmid, pSV2cat. This expression level in B-cells was further diminished by the inclusion of an immunoglobulin enhancer in the DT-A plasmid. In cotransfection experiments with a DT-A plasmid lacking an enhancer, chloramphenicol acetyltransferase expression was much more strongly inhibited in 293 cells (which express adenovirus E1A and E1B products) than in the other cell types; furthermore, the presence of the DT-A sequence eliminated recovery of G418-resistant 293 cell transformants after transfection with a plasmid containing the neo selectable marker. These results suggest that cell-specific regulatory mechanisms can be exploited to achieve selective cell killing by expression of an introduced toxin gene.
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PMID:Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide. 346 Jun 97

Downregulation of the fibronectin (FN) gene in a rat 3Y1 derivative cell line, XhoC, transformed by the adenovirus E1A and E1B genes seems to be caused by the induction of a negative regulator, G10BP, which binds to three G-rich sequences in the promoter (T. Nakamura, T. Nakajima, S. Tsunoda, S. Nakada, K. Oda, H. Tsurui, and A. Wada, J. Virol. 66:6436-6450, 1992). These are the G10 stretch and two GC boxes consisting of the G10 stretch with one internal C residue insertion. The recognition sequences of G10BP and Sp1 (GGGCGG) overlap in these GC boxes. To analyze the mechanism of the downregulation, G10BP was purified by DNA affinity chromatography, and its molecular mass was estimated to be about 30 kDa. The promoter was modified by substituting the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, and by replacing the Sp1 motif by the T stretch. Transcription of FN promoter-chloramphenicol acetyltransferase fusion genes carrying the base substitution in one or more of these G-rich sequences both in vivo and in vitro revealed that the base substitution in any G-rich sequence results in reduction of promoter activity, although the downstream GC box (GCd) plays a primary role. The addition of G10BP severely inhibited the activities of the FN promoters carrying the wild-type GCd in vitro, while the promoters carrying the mutant GCd were unaffected. The binding affinity of G10BP and Sp1 to each of the G-rich sequences, analyzed by gel shift assays, indicated that G10BP binds strongly to the GCd, moderately to the G10 stretch, and weakly to GCu, while Sp1 binds strongly to GCu, moderately to GCd, and weakly to the G10 stretch. Sp1 binding to GCd and the G10 stretch was inhibited by G10BP, while binding to GCu was unaffected. These results indicate that FN gene transcription is inhibited in XhoC cells primarily by exclusion of Sp1 binding to GCd by G10BP and that G10BP is a new class of Sp1 negative regulator.
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PMID:G10BP, an E1A-inducible negative regulator of Sp1, represses transcription of the rat fibronectin gene. 756 93

To explore the role of the E7 viral oncogene from human papillomavirus type 16 (HPV 16) in the regulation of cytoskeletal organization, we investigated alterations in particular cytoskeletal components in rat embryonal fibroblasts and three transformants of rat embryonal fibroblast cells produced by transfections with HPV16 E7 alone (TF1), HPV16 E7 plus adenovirus type 5 E1B (TF3), and HPV16 E7 plus activated Ha-ras (TF4). Marked reductions in smooth-muscle (SM) alpha-actin content and disrupted organization of stress fibers detected by anti-SM alpha-actin antibody were evident in all the transformants. These cytoskeletal manifestations were associated with a significant reduction in the mRNAs in these cells. Transcriptional repression by the E7 gene was observed after transient transfection of a chloramphenicol acetyltransferase reporter gene with SM alpha-actin gene promoter. Nucleotides -123 to -39 of the SM alpha-actin gene promoter were required for the HPV16 E7 transcriptional repression as shown by the chloramphenicol acetyltransferase assay. The downregulation of this actin isoform mediated by the E7 oncoprotein may play an important role in cell transformation by HPV16.
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PMID:Transcriptional repression of smooth-muscle alpha-actin gene associated with human papillomavirus type 16 E7 expression. 761 18


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