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)

Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Behavior of genes directly injected into the rat heart in vivo. 130 14

LINE-1 (L1) is a retroposon found in all mammals. In the mouse, approximately 10% of L1 elements are full-length and can be grouped into two classes, A or F, based upon the type of monomer sequence repeated at the 5' end. In order to test for promoter activity in the 5' end of the A-type mouse L1 element, we cloned several different A-monomers into a promoterless chloramphenicol acetyltransferase (CAT) vector. The A-monomer constructs varied in their ability to regulate transcription of the CAT gene, exhibiting CAT activity 16-37% of that detected with the Rous sarcoma virus promoter and enhancer. A series of A-monomer deletions were tested for their ability to regulate CAT expression and gel retardation experiments were performed to identify regions of the A-monomer that may be involved in L1 transcriptional regulation. A-monomer sequences are usually found repeated 2-5 times at the 5' end of a full-length mouse L1. In the absence of long terminal repeats or an internal promoter, the tandem array of A-monomers may provide a mechanism for A-type L1 elements to generate transcripts containing transcriptional regulatory sequences.
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PMID:Identification of transcriptional regulatory activity within the 5' A-type monomer sequence of the mouse LINE-1 retroposon. 131 70

We describe conditions under which exogenous DNA templates can be introduced for transient expression into primary murine T lymphocytes. T cells at various stages of development, including concanavalin A-activated splenic T cells, immature pre-T cells, and even small cortical thymocytes, could be successfully transfected. A variety of model DNA constructs were compared in which different viral promoter regions were used to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene. All showed enhanced expression in cells that had been acutely stimulated with the Ca2+ ionophore A23187 and phorbol ester as chemical proxies for T-cell receptor-mediated signals. In addition, splenocytes but not thymocytes required prior treatment with a mitogen and interleukin-2 in order to express these constructs, implying that even postmitotic thymocytes may be held in a quasiactivated state. A most striking result was the finding that the viral regulatory sequences in the Rous sarcoma virus long terminal repeat and the simian virus 40 early region were subject to sharply differential regulation, with a rank order that changed depending on the developmental stage of the T cells. The most immature thymic blasts and several lymphoma cell lines expressed the pRSV-Cat and pSV2-Cat constructs similarly, but cortical thymocytes exhibited a strong preference for pSV2-Cat. Splenic concanavalin A-stimulated blasts, on the other hand, slightly preferred pRSV-Cat, a tendency which became exaggerated in factor-dependent T-cell lines. The ratio of pRSV-Cat to pSV2-Cat expression varied according to cell type by as much as 500-fold. These results argue against a trivial linkage of promoter preference to cell cycle status but instead provide evidence that activation of T cells at distinct stages of differentiation results in the expression of different ensembles of nuclear regulatory proteins. In contrast to the simian virus 40 and Rous sarcoma virus promoter regions, the long terminal repeats of the retroviruses mink cell focus-forming virus and Akv were expressed well in all primary T-lineage cells. Thus, they represent excellent model promoters for engineering developmental stage-independent expression of exogenous genes in murine T cells.
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PMID:In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters. 131 65

Northern-blot analysis was used to demonstrate that an increase in extracellular glucose concentration increased the content of preproinsulin mRNA 2.3-fold in the beta-cell line HIT T15. A probe for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase was used as a control. Mannoheptulose blocked this effect of glucose. A stimulatory effect on preproinsulin mRNA levels was also observed in response to mannose and to 4-methyl-2-oxopentanoate. However, galactose and arginine were ineffective. Glucagon, forskolin and dibutyryl cyclic AMP also elicited an increase in HIT-cell preproinsulin mRNA. The ability of the 5' upstream region of the preproinsulin gene to mediate the effect of glucose and other metabolites on transcription was studied by using a bacterial reporter gene technique. HIT cells were transfected with a plasmid, pOK1, containing the upstream region of the rat insulin-1 gene (-345 to +1) linked to chloramphenicol acetyltransferase (CAT). Co-transfection with a plasmid pRSV beta-gal containing beta-galactosidase driven by the Rous sarcoma virus promoter was used as a control for the efficiency of transfection; expression of CAT activity in transfected HIT cells was normalized by reference to expression of beta-galactosidase. Glucose caused a dose-dependent increase in expression of CAT activity, with a half-maximal effect at 5.5 mM and a maximum response of 4-fold. Mannoheptulose blocked this effect of glucose. Other metabolites (mannose, 4-methyl-2-oxopentanoate and leucine plus glutamine) were also able to increase insulin promoter-driven CAT expression, but galactose and arginine were ineffective. The stimulatory effect of glucose on CAT expression was not blocked by verapamil and was inhibited by increasing extracellular Ca2+ from 0.4 to 5 mM. Both dibutyryl cyclic AMP and forskolin caused an increase in insulin promoter-driven gene expression in the presence of 1 mM-glucose, but neither agent further increased the level of expression occurring in the presence of a maximally stimulating glucose concentration. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also increased insulin promoter-driven CAT expression in the presence of 1 mM-, but not 11 mM-glucose. Staurosporine blocked the stimulatory effect not only of PMA but also of glucose and of dibutyryl cyclic AMP. We conclude that the 5' upstream region of the insulin gene contains sequences responsible for mediating the stimulatory effect of glucose on insulin-gene transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Control of insulin gene expression by glucose. 132 37

We report the fish use of an exponential decay electroporation system to introduce foreign DNA into fertilized zebrafish embryos. The plasmid RSVCAT (Rous sarcoma viral promoter (RSV) upstream from the chloramphenicol acetyltransferase gene (CAT)) was linearized and introduced into fertile zebrafish embryos by electroporation no later than the four-cell stage. Conditions for the procedure were empirically derived, and 68% of the treated animals survived through hatching to at least 6 days after fertilization and well beyond. Dot-blot analysis on DNA extracted from individual hatching fry demonstrated that 65% of the animals tested carried the foreign construct. Enzyme assays on the soluble proteins of treated animals were positive for chloramphenicol acetyltransferase activity. These data demonstrate that the foreign construct was being transiently expressed in the developing tissues of the embryo. The simplicity of this technique will greatly enhance the ability to analyze gene promoter regulation in vivo in transgenic zebrafish. The ability of the electroporated DNA to integrate into the host genome and to generate stable lines of transgenic fish is discussed.
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PMID:Transient expression of RSVCAT in transgenic zebrafish made by electroporation. 133 27

Mutation of the p53 tumor suppressor gene is a recurring event in a variety of human cancers. Wild-type p53 may regulate cell proliferation and has recently been shown to repress transcription from several cellular promoters. We studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen promoter and on several viral promoters including the simian virus 40 early promoter-enhancer, the herpes simplex virus type 1 thymidine kinase and UL9 promoters, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus, human immunodeficiency virus type 1, and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and plasmids containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. Expression of wild-type p53 correlated with a consistent and significant (6- to 76-fold) reduction of reporter enzyme activity. A mutation at amino acid 143 of p53 releases this inhibition significantly with all the promoters studied. Expression of a p53 mutated at any one of the five amino acid positions 143, 175, 248, 273, and 281 also correlated with a much smaller (one- to sixfold) reduction of reporter enzyme activity from the herpes simplex virus type 1 thymidine kinase promoter. These mutant forms of p53 are found in various cancer cells. Thus, failure of tumor suppression correlates with loss of the promoter inhibitory effect of p53.
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PMID:Inhibition of viral and cellular promoters by human wild-type p53. 135 31

Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.
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PMID:Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells. 135 62

The H-2Kb gene equipped with a minimal promoter (5' deletion up to -61) was fully expressed in transfected fibroblasts, but inactive in transfected embryonal carcinoma cells. A strong transcriptional regulatory element (H2DRE) was identified when a fragment spanning the second exon and second intron was used to activate transient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in mouse Ltk- or NIH3T3 fibroblasts. Its activity was twice that of a construct where the CAT gene was driven by the H-2Kb 5' enhancer region (H2TF1/KBF1 site) and comparable to that of pRSVCAT construct carrying the strong Rous sarcoma virus LTR enhancer. In accord with regulated transcriptional activity of the intact H-2Kb gene, the H2DRE did not activate the CAT expression in P19 mouse embryonal carcinoma cells. The H2DRE did not function as a typical enhancer since its activity was strongly position dependent. Consistent with its anticipated role in transcription regulation, H2DRE displayed more than five target sites for specifically interacting nuclear factors, two of them being present in H-2 positive fibroblasts, but not in H-2 negative teratocarcinoma cells. None of them was cross-competed by sequences of the 5' enhancer. The results of deletion experiments show that H2DRE is the only regulatory region that can activate transcription from the 5' enhancerless H-2Kb gene in mouse L fibroblasts.
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PMID:A novel downstream regulatory element of the mouse H-2Kb class I major histocompatibility gene. 142 92

We have isolated the avian gene T64 corresponding to the mammalian clusterin, on the basis of high accumulation of its template mRNA in cells infected with oncogenic retroviruses. Since the clusterin was shown to have a protective effect against the immune system, its induction by oncogenic viruses is of major biological importance. The unique, short 5 kb-long T64 genomic locus is inactive in normal quail embryo fibroblasts in primary culture whereas it shows a high transcriptional activity after transformation by the Rous sarcoma virus. The 963 bp-long 5' flanking region is sufficient to drive the transcription of the chloramphenicol acetyltransferase reporter gene in a thermodependent manner when a thermosensitive version of pp60v-src is used. Deletion and point mutation analyses of the promoter show that the v-src response requires at least two separate elements: PUR and AP-1, located respectively at positions -167 to -152 and -25 to -19 relative to the single transcription initiation site. In addition, the binding of specific nuclear factors to these responsive elements correlates with the T64 promoter activation.
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PMID:V-src-induced-transcription of the avian clusterin gene. 147 99

Quail myogenic cells infected with temperature sensitive (ts) mutants of Rous sarcoma virus (RSV) exhibit a temperature-dependent transformation and block of differentiation. When the cells are allowed to differentiate at the restrictive temperature (41 degrees C) and then shifted back to the permissive temperature (35 degrees C), a sharp reduction in the accumulation of muscle-specific mRNAs is observed, following reactivation of the transforming protein pp60v-src. A kinetic analysis of this down-regulation reveals that the reduction in the accumulation of muscle-specific transcripts occurs fairly rapidly within 6 to 20 h after the shift back, depending on the mRNA analyzed. Studies on transcription of endogenous muscle-specific genes and a transfected chloramphenicol acetyltransferase reporter gene under the control of muscle-specific promoters, at the different temperatures, suggest that the oncogene exerts its control mainly at the transcriptional level. On the contrary, transcription of the CMD1 gene, the avian homolog of the mouse muscle regulatory MyoD gene, is not significantly affected by the oncogene both in proliferating myoblasts and in myotubes shifted back to 35 degrees C. These findings are consistent with the conclusion that v-src blocks myogenesis by controlling transcription of muscle-specific genes independently of cell proliferation. Furthermore, they suggest the existence of an alternative pathway, not requiring the silencing of CMD1 transcription, through which the oncogene exerts its effect.
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PMID:Transcription of muscle-specific genes is repressed by reactivation of pp60v-src in postmitotic quail myotubes. 164 48


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