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)

Murine embryonal carcinoma F9 cells, which do not express appreciable levels of major histocompatibility complex (MHC) class I mRNA, start to express the mRNA and proteins upon differentiation induced by retinoic acid (RA). To investigate the molecular mechanism of this regulation, we examined in F9 cells transient expression of the chloramphenicol acetyltransferase (CAT) gene directed by the 5' flanking region of a MHC class I gene, H-2Ld. The native 1.4-kilobase H-2Ld 5' upstream region gave very low CAT activity in undifferentiated F9 cells. Deletion between positions -210 and -135 relative to the cap site resulted in a 4- to 5-fold increase in CAT activity as compared with constructs containing the region. However, all of these constructs, regardless of the deletion, expressed comparable CAT activity in differentiated F9 cells. These data suggest the presence of a negative cis-acting element that is under developmental control. Further analysis revealed that the sequence conferring the negative regulation resides between positions -195 and -161. This region, highly conserved among the MHC class I genes, is found to be capable of increasing CAT activity in NIH 3T3 cells that express the class I genes constitutively. Further, this regulatory sequence, when connected to the simian virus 40 promoter, produced repressive and enhancing effects in F9 and NIH 3T3 cells, respectively. Based on these results, we suggest that the expression of MHC class I genes during development involves switching from negative to positive regulation dictated by the class I regulatory element located between positions -195 and -161.
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PMID:Negative regulation of the major histocompatibility class I gene in undifferentiated embryonal carcinoma cells. 346 24

The 5'-upstream region of the rat alpha-fetoprotein (AFP) gene strongly increased de novo methylation of an adjacent chloramphenicol acetyltransferase (CAT) gene upon transfection into F9 mouse embryonal carcinoma cells. The same effect was exerted by a distal 775-base pair (bp) fragment and by 300- and 1-kb fragments preceding the transcriptional start site, but not by other parts of the control region. Further division of the larger, strongly active fragments resulted in a gradual decrease of methylation and clonal variation in the methylation patterns. The effect of the 775-bp fragment did not depend on its orientation. It was ablated by insertion of the mouse metallothionein I promoter between the AFP gene fragment and the CAT gene, but not by its insertion upstream of the AFP gene fragment. Two fragments from the AFP control region increasing methylation contained B1 and B2 small interspersed repetitive elements, respectively. B1 and B2 sequences of different origin also acted strongly to increase methylation. These findings support the idea that mammalian genes contain specific sequences involved in regulating their methylation. The effects of these sequences appear to be exerted in cis, to be dependent on proximity, but not on orientation, and to require an optimal size of 500-700 bp. Small retrotransposon sequences within such elements may be particularly effective in attracting de novo methylation.
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PMID:Enhancement of reporter gene de novo methylation by DNA fragments from the alpha-fetoprotein control region. 750 85

We previously isolated a cDNA clone encoding interferon consensus sequence-binding protein (ICSBP), a member of the interferon regulatory factor (IRF) family, that binds to the interferon (IFN)-stimulated response element (ISRE) of many IFN-regulated genes. In this investigation, we studied the functional role of ICSBP by transient cotransfection of ICSBP cDNA with IFN-responsive reporter genes into the human embryonal carcinoma cell line N-Tera2. These cells were shown not to express ICSBP or IRF-2, thus allowing functional analysis of transfected cDNAs. Cotransfection of ICSBP into cells treated with retinoic acid or any of the IFNs (alpha, beta, or gamma) repressed expression of a chloramphenicol acetyltransferase reporter driven by the major histocompatibility complex class I gene promoter. Similarly, ICSBP repressed expression of chloramphenicol acetyltransferase reporters driven by the ISREs of the 2'-5' oligoadenylate synthetase, guanylate-binding protein, and ISG-15 genes in IFN-treated cells. The repression was dependent on the presence of the ISRE in the reporter. Deletion analysis showed that the putative N-terminal DNA binding domain of ICSBP by itself is capable of mediating the repression. Using the same cotransfection conditions as for ICSBP, a similar repression of these reporters was observed with IRF-2. Finally, ICSBP repressed the IRF-1-mediated induction of major histocompatibility complex class I and IFN-beta reporters in the absence of IFN or retinoic acid. Taken together, these results suggest that ICSBP is a negative regulatory factor capable of repressing transcription of target genes induced by IFN, retinoic acid, or IRF-1.
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PMID:Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferon-induced gene transcription. 767 54

BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct revealed that the transcription of BOXF1 gene is regulated by BOX DNA, preferentially in undifferentiated EC cells versus differentiated cells. Hence, BOX DNA is probably a novel transcriptional element related to EC cell differentiation.
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PMID:BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation. 790 32

Expression of hst (k-FGF, FGF-4), a member of the fibroblast growth factor gene family, is restricted to early stages of developing embryos and to embryonal carcinoma cells. In F9, which is a prototype of embryonal carcinoma cells expressing hst, the expression of hst gene is positively regulated by a downstream octamer motif that functions as an enhancer. We have investigated, by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in F9, the cis-acting regulatory element within the hst promoter region that interacts with this enhancer. Electrophoretic mobility shift assay and methylation interference analysis showed that the hst promoter contains, in a segment termed Y, the sequence 5'-CTGATTGGCA-3', which closely resembles the consensus binding motif for the CCAAT-binding factor NF-Y. Deletions or mutations in this element substantially reduced expression of hst-CAT constructs. The nuclear factor binding to the Y segment of the hst promoter was indistinguishable from NF-Y, as inferred from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. We conclude that the expression of the hst gene in F9 is positively regulated by the coordinated interaction between an NF-Y-binding site and an octamer motif.
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PMID:An upstream NF-Y-binding site is required for transcriptional activation from the hst promoter in F9 embryonal carcinoma cells. 792 90

The majority of cellular mRNAs have relatively short and unstructured 5' untranslated regions (UTRs) that allow efficient translation, such as the beta-globin mRNA. An exception to this rule is the group of growth factor mRNAs which, in general, have long 5' UTRs with a high G + C content. An example is insulin-like growth factor II (IGF-II), which is encoded by four mRNAs, arising from four different promoters. Transcripts having the human IGF-II leader 1 are only expressed in adult liver where IGF-II protein synthesis is solely under direction of this 5' UTR. We investigated the translational efficiency in vitro of this 5' UTR, linked to the chloramphenicol acetyltransferase (CAT) encoding region. As expected from the primary structure of IGF-II leader 1, translational efficiency was very low compared with beta-globin 5' UTR-CAT mRNA. Addition of cell extract from undifferentiated P19 embryonal carcinoma (EC) cells preferentially stimulated translation of an IGF-II 5' UTR RNA construct. No translational stimulation was found when cell extract from differentiated P19 EC cells was added. In contrast with the beta-globin 5' UTR, translation initiation on the IGF-II 5' UTR was not dependent on the presence of a cap structure. The results imply that only in undifferentiated P19 EC cells and not in their differentiated derivatives is a factor present that specifically stimulates IGF-II RNA translation, thereby suggesting translational regulation of IGF-II production during early embryonic development. A mechanism for translation initiation on the 5' UTR of IGF-II is discussed.
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PMID:Translation initiation on the insulin-like growth factor II leader 1 is developmentally regulated. 798 Apr 16

The molecular mechanisms by which expression of a gene is down-regulated after differentiation of F9 embryonal carcinoma cells into parietal endoderm-like cells was studied by characterizing the cis- and trans-regulatory elements of the gb110 gene. This gene encodes a putative RNA helicase, and its expression is down-regulated when F9 cells are differentiated with retinoic acid and cyclic AMP. The 5'-flanking region of the gene has all of the features of a GC-rich island promoter and seems to play only a minor role, if any, in the regulated expression. A 133-bp enhancer in the first intron was identified by transient chloramphenicol acetyltransferase assays that activated expression in undifferentiated F9 cells about 50- to 100-fold. As this enhancer was not active in differentiated F9 cells, it seems to be the prime mediator of the differentiation-specific down-regulation of the gb110 gene. Four different protein-binding sites, three of which contain GC- and GT-box motifs, were identified in the enhancer element. The fourth site, interacting with previously described transcription factor FTZ-F1/ELP, seems to be of minor importance for the activity of the enhancer. Mutational analysis showed that the cooperative interaction of several most likely related proteins with the three GC- and GT-box motifs was required for full enhancer activity. On the basis of their binding properties, at least two of these proteins seem to be identical or closely related to ubiquitous transcription factor Sp1. One of the GT-box-binding proteins was present in undifferentiated F9 cells but not, however, in its differentiated derivatives. The cell specificity of this transcription factor explains why the gb110 gene is not expressed or expressed only at low levels in parietal endoderm-like cells.
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PMID:Interaction of several related GC-box- and GT-box-binding proteins with the intronic enhancer is required for differential expression of the gb110 gene in embryonal carcinoma cells. 806 13

We evaluated SR11237, a retinoid X receptor (RXR)-specific compound, for its pharmacologic effects on cell differentiation in F9 embryonal carcinoma cells and rhino mouse epidermis. SR11237 can cause RXR/RXR homodimers to form and transactivate a reporter gene containing a RXR-response element. We confirmed, using nuclear receptor co-transfection assays in COS-1 cells, that SR11237 is effective at transactivating a chloramphenicol acetyltransferase reporter gene through RXRs but not retinoic acid receptors. When SR11237 was tested for its ability to modulate cell differentiation, it was inactive on F9 embryonal carcinoma cells and rhino mouse skin. Because differentiation in these systems is known to be regulated by RAR-specific compounds, such as all-trans-retinoic acid and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope nyl benzoic acid], our results with SR11237 are compatible with the concept that classical retinoid pleiotropic responses are mediated by RXR/RAR heterodimeric nuclear receptors rather than through RXR/RXR homodimers.
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PMID:A pleiotropic response is induced in F9 embryonal carcinoma cells and rhino mouse skin by All-trans-retinoic acid, a RAR agonist but not by SR11237, a RXR-selective agonist. 817 47

Changes in the neuronal content of neurofilament proteins occur in some neuropathological conditions, but little is known about the molecular mechanisms that control both the cell type specificity and the levels of expression of neurofilament genes. In addition to TATA and Sp1 elements, we report here the presence in the neurofilament light (NF-L) promoter region of other regulatory elements, namely, an AP-1 element TGCGTCAG, a Krox-24 element GCACCCCGC, and an Ets-like element AGCAAGCAGGAATTT. These elements constitute binding sites for specific nuclear factors present in aggregated P19 embryonal carcinoma cells. Using cotransfection assays in P19 embryonal carcinoma cells, we show that NF-L promoter fragments fused to the reporter chloramphenicol acetyltransferase gene can be trans-activated by expression vectors encoding FOS and JUN (AP-1) and by Krox-24 protein. The finding of functional elements for immediate early gene products in the NF-L promoter suggests molecular pathways by which the modulation of neurofilament expression can be coupled to growth factors and other external stimuli.
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PMID:AP-1 and Krox-24 transcription factors activate the neurofilament light gene promoter in P19 embryonal carcinoma cells. 818 Jan 32

We reported that a cell surface thrombin receptor, thrombomodulin (TM), was regulated by cyclic AMP in fibroblasts and in parietal endoderm-like cells derived from F9 embryonal carcinoma cells. In this paper, the genetic basis for augmentation of TM expression by cyclic AMP was studied in F9 and BALB/3T3 cells. Transient expression assays were performed with plasmid constructs containing various 5' flanking sequences of the TM gene and a reporter gene, chloramphenicol acetyltransferase (CAT). Two regulatory DNA regions, the proximal (-411 to -50) and the distal (-1026 to -850), were located. Interplay of the two regions was suggested using a heterologous thymidine kinase promoter in differentiated F9 cells. Both proximal and distal regions contributed to cyclic AMP-dependent augmentation of CAT expression in differentiated F9 cells, whereas only the proximal region was functional in BALB/3T3 cells. The two cell types responded differently also to a protein synthesis inhibitor, cycloheximide, with respect to TM message accumulation. In BALB/3T3 cells TM message accumulation was refractory to the inhibitor in contrast to that of differentiated F9 cells, which was only partially so. We propose that there are at least two separate genomic DNA regions that regulate cyclic AMP-dependent TM gene expression and that their functions are cell type dependent.
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PMID:Cyclic AMP-mediated augmentation of thrombomodulin gene expression: cell type-dependent usage of control regions. 839 1


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