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)

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.
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PMID:Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies. 246 54

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was cotransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.
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PMID:Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation. 253 33

The long terminal repeats (LTRs) of the human immunodeficiency virus (HIV) the Rous sarcoma virus (RSV) and the copia Drosophila retrotransposon were compared in their capacity to direct expression of the bacterial cat (chloramphenicol acetyltransferase) gene in human, murine, and Drosophila cell lines. The results indicate that HIV and RSV LTR expression is post transcriptionally repressed in the Drosophila cells while copia LTR expression is post-transcriptionally repressed in the human and murine cells.
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PMID:HIV-1 expression is posttranscriptionally repressed in Drosophila cells. 254 51

Transfection of cultured cells with functioning foreign genes can be a powerful tool for delineating mechanisms controlling gene expression and for manipulating cellular synthetic machinery. We transfected cultured bovine pulmonary artery endothelial cells with a plasmid (pRSVCAT) containing the prokaryotic gene, chloramphenicol acetyltransferase (CAT), driven by a Rous sarcoma virus (RSV) promoter. Transfection was accomplished by binding the plasmid DNA to specially synthesized cationic liposomes and incubating cell monolayers with the liposome-DNA complex (a process called lipofection). We found marked CAT expression in endothelial cells by 24 h after lipofection (complete chloramphenicol acetylation in a 4-h assay incubation with less than 0.1 mg cell protein). Marked CAT expression persisted after splitting the cells 1:2 at 6 days after lipofection. Detectable CAT activity was present 14 days after lipofection following two 1:2 splits of the cells. CAT expression was related to the quantity of plasmid DNA used for lipofection; 1.0 micrograms DNA per 60-mm dish of cells resulted in less CAT activity at 48 h than did 5 or 10 micrograms DNA per dish. Ten micrograms of DNA-liposome complex per dish caused substantial numbers of endothelial cells to detach from the dish, but little cytotoxic effect was seen with 5 or 1 micrograms DNA-liposome complex per dish. This simple, highly efficient method for introducing functioning foreign genes into cultured endothelial cells will permit a broad range of studies of molecular mechanisms of endothelial cell function and studies of the consequences of highly specific modifications in protein synthesis on endothelial responses.
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PMID:Expression of a prokaryotic gene in cultured lung endothelial cells after lipofection with a plasmid vector. 255 63

Eight eukaryotic promoters have been tested for their activity in vivo in Escherichia coli. The rat beta-actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) and hepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by primer extension RNA analysis. All eight promoter-chloramphenicol acetyltransferase constructs produce chloramphenicol acetyltransferase activity with the following relative strengths: RSV LTR greater than rat beta-actin greater than rat insulin I greater than rat amylase greater than hepatitis B virus precore greater than human insulin greater than rat chymotrypsin B greater than mouse metallothionein I. A primer extension analysis indicates that transcription from the RSV LTR, rat insulin I, and rat beta-actin promoters initiates at the sites expected for eukaryotic rather than prokaryotic promoters. Thus the site of initiation is determined by the DNA sequence rather than by the RNA polymerase.
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PMID:Eukaryotic promoters drive gene expression in Escherichia coli. 268 Nov 82

In preparation for studies using gene transfer, we have identified transcriptional control elements which are active in primary rat hepatocytes. We used plasmids which were constructed so that the promoter or enhancer of interest initiated transcription of the bacterial chloramphenicol acetyltransferase (CAT) gene. Plasmids were introduced into primary rat hepatocytes in culture, into Hep G2 cells and other human and animal cell lines and into bone marrow stromal cells, and CAT activity was assayed after 48 hr. In primary rat hepatocytes, the highest CAT activity was obtained with plasmids carrying the Rous sarcoma virus long terminal repeat (pRSVCAT), or the SV40 early region promoter and enhancer (pSV2CAT). Hepatocytes carrying the murine cytomegalovirus immediate early promoter (pUCRNmCMVX/HCAT) also had appreciable CAT activity. No CAT activity was detected in rat hepatocytes carrying pSVOCAT (a promoterless construct), pUCRNtKCAT (herpes simplex thymidine kinase gene promoter), pLPVCAT (lymphocytotrophic papovavirus promoter) and pHBV1CAT (hepatitis B virus enhancer and core gene promoter). Therefore, for future studies of gene transfer in primary rat hepatocytes, the Rous sarcoma virus long terminal repeat or the SV40 early region promoter and enhancer can be effectively used to drive gene expression. Hep G2 cells carrying pHBV1CAT had high CAT activity. Hep G2 cells carrying pHBV2CAT (similar to pHBV1CAT, but with the hepatitis B virus sequences in reverse orientation with respect to the CAT sequences) and pHBV3CAT (similar to pHBV2CAT, but hepatitis B virus sequences are separated from the CAT sequences by about 700 bases) also expressed CAT activity, but not as strongly as with pHBV1CAT.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue-specific activity of heterologous viral promoters in primary rat hepatocytes and Hep G2 cells. 280 56

The ability of hepatitis B virus (HBV) to stimulate the expression of a cellular gene was investigated by using a transient-expression system. A plasmid in which the expression of the bacterial chloramphenicol acetyltransferase (cat) gene had been placed under the control of the DNA sequences that regulate the expression of the human beta-interferon gene was constructed. In Vero cells, cotransfection of the 2.7-kilobase BglII DNA fragment of HBV together with the test plasmid containing the cat gene resulted in stimulation of the expression of the cat gene. This HBV DNA fragment was specific in its trans-activation; no significant stimulation of CAT activity was observed in constructs when the promoter and enhancer elements were derived from the murine sarcoma viral long terminal repeat, Rous sarcoma virus, BK virus, or simian virus 40. Results of subcloning of the HBV DNA fragment indicate that the trans-activating function resides in a 944-base-pair EcoRV-BglII DNA fragment of the HBV genome that contains the X structural gene and its promoter element. Removal of the promoter from the X structural gene resulted in loss of the trans-activating function. A frameshift mutation within the X gene region also eliminated the trans-activating activity. These results suggest that the X antigen could play a role in HBV infections by activating the expression of cellular genes.
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PMID:Transcriptional trans-activating function of hepatitis B virus. 282 53

We have used the chloramphenicol acetyltransferase (cat) gene expression system to study the effect of the X protein of hepatitis B virus (HBV) on viral enhancers. Plasmids containing the HBV enhancer and the core gene promoter linked to the cat gene were cotransfected with a plasmid containing the X gene into the human hepatoma cell line PLC/PRF/5. Our results indicate that the transfected X gene caused a trans-activation of the HBV enhancer. If a frameshift mutation or a deletion in the X structural gene was created, this trans-activation function was abolished. This result and the observation that the frameshift mutation did not alter the transcription of X mRNA suggest that the X protein is the trans-activating factor. Using similar techniques, we found that the X protein was also capable of trans-activating the simian virus 40 (SV40) and Rous sarcoma virus enhancers (pSV2cat and pRSVcat) in CV-1 cells. However, trans-activation of the SV40 enhancer by the X protein was not observed in COS-1 cells. By cotransfecting pSV2cat and the X gene with a plasmid containing either the intact SV40 genome, the SV40 genome devoid of the T-antigen (T-ag) gene, or only the T-ag gene, we demonstrated that SV40 T-ag can suppress trans-activation by the X protein. SV40 T-ag did not inhibit expression of the X gene or inactivate the X protein. The most probable mechanism of this inhibition is that T-ag competes with the X protein for a common target.
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PMID:trans-activation of viral enhancers by the hepatitis B virus X protein. 282 5

We have produced a line of transgenic mice that is characterized by prenatal lethality. These mice bear a chimeric plasmid containing the long terminal repeat of the Rous sarcoma virus linked to the coding region of the chloramphenicol acetyltransferase gene (pRSV-CAT). Mice heterozygous for the pRSV-CAT integration site are semisterile, producing litters approximately equal to 40% of the average size when crossed to normal mice. Approximately 50% of the progeny from such a cross bear the pRSV-CAT sequences and also produce litters of smaller size. An analysis of embryogenesis revealed that normal numbers of embryos implanted, but 60% failed to develop past day 7. Eight other independent transgenic lines containing RSV-CAT show no evidence of embryonic lethality; thus, it is unlikely that the defect observed is due to the direct effects of RSV-CAT expression. We have found that carrier mice bear a reciprocal translocation between chromosomes 6 and 17, T(6A2-6A3;17D-17E1), that can explain the apparent dominant embryonic lethality seen in this line. The site of integration has been localized by in situ hybridization at or near the translocation breakpoint in one of the translocated chromosomes (6(17)). Because the foreign DNA is present in one of the translocated chromosomes, we propose that this rearrangement was elicited by the introduction of foreign DNA.
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PMID:Prenatal lethality in a transgenic mouse line is the result of a chromosomal translocation. 282 18

We investigated the role of sequences flanking the transcription initiation site of the delta 1-crystallin gene in transient transfection assays of primary embryonic chicken lens epithelial cells or fibroblasts. Varying lengths of the 5' flanking sequence of the delta 1-crystallin gene (containing some untranslated sequence from exon 1) were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene in the pSVOCAT plasmid. A plasmid carrying the bacterial beta-galactosidase gene driven by the Rous sarcoma virus (RSV) promoter was used as an internal control. Standardized results showed that the sequence located between -120 to -43 exhibited strong promoter activity; however, the promoter activity was markedly reduced (20-fold) when the upstream sequence between -603 and -120 was included in the construct. The delta 1-crystallin promoter displayed little lens preference. This upstream sequence did not reduce the activity of the Simian virus 40 (SV40) early promoter (with or without its enhancer) or the Herpes thymidine kinase promoter in transfection tests, indicating some specificity in its effect. Evidence for a delta 1-crystallin negative trans-acting factor was provided by competition experiments. Our data raise the possibility that expression of the delta 1-crystallin gene involves a negative cis-acting transcription element, a speculation which may deserve further attention in view of the gradual decrease in delta-crystallin synthesis in the developing lens.
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PMID:Evidence for positive and negative regulation in the promoter of the chicken delta 1-crystallin gene. 283 46


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