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)

In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.
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PMID:Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes. 867 28

This report describes the use of restriction enzyme-mediated integration (REMI) to increase the transformation frequency and allow co-transfection of several unselected constructs under the selection of a single selectable marker. We found that while BamHI (the enzyme used to originally demonstrate REMI (Schiestl, R.H. and Petes, T.D. (1991) Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Nati. Acad. Sci. USA 88, 7585-7589) increased the number of transformants by 2-5-fold over the control without added enzyme, NotI proved to be a further 29-46-times more effective in enhancing stable transformation. This simple technique was used in the transformation of three non-selective markers (two modified membrane proteins and beta-galactosidase) with a selectable construct expressing chloramphenicol acetyltransferase. Following chloramphenicol selection, four out of ten independent transformants stably acquired all four constructs with at least two expressing all four genes at the protein level. These results demonstrate that REMI may be used in the efficient stable transformation and co-transfection of this and perhaps other protozoan parasites.
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PMID:Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii. 871 45

In this article we describe an improved method to produce a conjugate of anti-erythrocyte growth factor (EGF) receptor monoclonal antibody with polylysine via thio-ether bonds. The resulting antibody/polylysine conjugate was found to be a much more stable DNA (gene) carrier than the previous conjugate formed via disulfide bonds. We designated the conjugate as an "immunoporter" and the immunoporter/DNA (gene) complex as an "immunogene." The fluorescent microscopic observation showed that the immunoporter as well as immunogene bound specifically to the EGF receptors on the cell surface, and the loaded reporter gene, such as beta-galactosidase (beta-GAL), was detected in the cell nucleus at 2 hours after transfection. The enzyme activity from the beta-GAL gene was detected at 12 hours and increased for 3 to 5 days. Similar kinetics were obtained for another reporter gene, chloramphenicol acetyltransferase. Furthermore, the immunoporter delivered the herpes simplex virus thymidine kinase gene and induced substantial suicide effects on tumor cells when gancyclovir or acyclovir was added. Thus, the immunogene approach was successful in delivering therapeutic genes to EGF receptor overexpressing tumor cells. Further technical refinement may prove useful as a supplementary treatment of patients with squamous cell carcinomas.
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PMID:Immunogene approach toward cancer therapy using erythrocyte growth factor receptor-mediated gene delivery. 872 10

Lipofection using the Lipofectin reagent was optimized to transiently transfect subcultured guinea pig endometrial stromal cells with a beta-galactosidase gene driven by a simian virus 40 promoter. Efficient transfection was obtained in the following conditions: a value of six for the ratio of lipofectin to DNA, a low cellular density (10(5) cells per 35-mm well) at the time of subculture (48 h before lipofection) and a lipofection duration of 12 hours. Lipofection was compared to calcium phosphate precipitation previously optimized in the same culture model. At a low cellular density, the lipofection method was found to be more efficient than the calcium phosphate precipitation. This result gives a great relevance to lipofection since the cultured cells available in an experiment are often limited. Then, using cells at low density and a plasmid containing the chloramphenicol acetyltransferase (cat) gene linked to an estrogen response element, it was shown that the lipofection procedure is a suitable tool for the evaluation of gene regulation by estrogen.
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PMID:Gene transfer into subcultured endometrial cells using lipofection. 877 Apr 11

The effects of mitogens and agents affecting tyrosine phosphorylation signaling on androgen-regulated transcription were investigated. CV-1 and HeLa cells were cotransfected with an androgen receptor (AR) expression vector and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse mammary tumor virus promoter. Growth factors [epidermal growth factor (EGF) and insulin-like growth factor I] that activate receptor tyrosine kinases, an inhibitor of phosphotyrosine phosphatases (vanadate), or an inhibitor of tyrosine kinases (genistein) did not influence basal promoter activity or that of unliganded AR. However, EGF, insulin-like growth factor I, and vanadate enhanced AR-dependent transactivation by 1.5- to 2.5-fold, and genistein diminished it by two thirds in the presence of androgen. None of the treatments affected pRSV-CAT or pSV-beta-galactosidase expression, suggesting that gross activation of the transcription machinery was not involved. A reporter with two androgen response elements (AREs) in front of the thymidine kinase promoter (p delta ARE2tk-CAT) was used to examine promoter specificity. EGF activated this reporter even in the absence of androgen. However, when EGF was used concomitantly with testosterone, it augmented the action of androgen. Vanadate enhanced androgen-induced transactivation 2-fold without altering basal promoter activity. Neither EGF nor vanadate altered immunoreactive AR content or elicited changes in the receptor's DNA-binding properties. The intracellular content of hormone-binding AR was not influenced by EGF, but was decreased by vanadate and increased by genistein, as judged by [3H]mibolerone binding assays. An AR form lacking the hormone-binding domain (delta 641-902 mutant) transactivated p delta ARE2tk-CAT reporter similar to or better than the wild-type receptor in the presence of androgen. The transactivation by the delta 641-902 mutant was augmented by EGF and vanadate, but was attenuated by genistein, implying that the steroid-binding region is not critical for regulatory events initiated by tyrosine phosphorylation. Collectively, these data indicate that there is cross-talk between androgen-mediated signaling systems and growth factor/receptor tyrosine kinase pathways.
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PMID:Effects of mitogens on androgen receptor-mediated transactivation. 882 95

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.
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PMID:A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung. 887 56

Several systems for the detection of HIV-1 have been described in which HIV-1-susceptible cells contain a reporter gene (chloramphenicol acetyltransferase, beta-galactosidase, or alkaline phosphatase) under the control of the HIV-1 long terminal repeat (LTR). Upon infection by HIV-1, the expression of the viral tat product increases transcription from the HIV-1 LTR promoter, leading to high-level expression of the reporter gene product. Previously described reporter systems require processing of the cells by lysis, fixation, or other steps following infection to detect the reporter gene product. In the present study, the Aequorea green fluorescent protein S65T variant (GFP-S65T) was used in a reporter system for detecting HIV-1. HeLa-CD4 cells transfected with the plasmid pRH1, which encodes GFP-S65T under the control of the HIV-1 LTR promoter, and either co-transfected with a plasmid encoding the HIV-1 tat product or superinfected with HIV-1, expressed high levels of GFP-S65T, which was readily detected by fluorescence microscopy and fluorescence-activated cell-sorting analysis. The advantages of this system include its simplicity, sensitivity, and ability to detect and sort live HIV-1-infected cells using readily available instruments. The construction of cell lines stably transfected with pRH1 will provide a tool for titering HIV-1 and sorting HIV-1-infected cells.
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PMID:Detection of HIV-1 infection with a green fluorescent protein reporter system. 894 67

Here, we report on the construction of three integrative plasmids for Bacillus subtilis (Bs) allowing in vitro construction of transcriptional fusions. These plasmids contain a neomycin- or tetracycline-resistance cassette and one of three promoterless genes: bgaB (encoding beta-galactosidase), cat (chloramphenicol acetyltransferase), or xylE (catechol 2,3-dioxygenase). All cassettes are flanked by the 3'- and 5'-ends of the amyE gene (encoding alpha-amylase) allowing integration of these cassettes at the amyE locus of the Bs chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the beta-lactamase-encoding bla gene. Four unique restriction sites can be used for insertion of restriction fragments carrying promoter fragments. All three reporter genes express heat-stable enzymes (stable up to at least 50 degrees C for 30 min) as shown here. We would like to point to the modular nature of these plasmids where the three reporter genes and the two resistance cassettes can be combined in any permutation. The versatility of the promoter-probe vectors was demonstrated by the integration of the promoters of the dnaK and groE operons of Bs and following their heat-inducible expression.
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PMID:Integrative vectors for constructing single-copy transcriptional fusions between Bacillus subtilis promoters and various reporter genes encoding heat-stable enzymes. 898 64

The use of viral vectors to deliver foreign genes offers some promise of generating new and more efficacious vaccines. However, the insertion of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affect replication. In an attempt to overcome this problem, we constructed two portable intron cassettes. The cassettes were derived from the adenovirus late leader 1 intron and were cloned into either the chloramphenicol acetyltransferase (CAT) gene or the LacZ gene of Escherichia coli. The intron cassettes were transfected into chicken embryo fibroblasts (CEFs) and the cell lysates were later assayed for either beta-galactosidase (beta-Gal) or CAT activity. The first intron cassette (type A) contained flanking adenovirus exon sequences. Consequently, the flanking adenovirus exon sequences remained in the spliced transcript. With the type A intron inserted in the correct orientation for splicing, CAT activity was not diminished. However, in the reverse orientation, no CAT activity could be detected. The second intron cassette (type B) had the splice donor and splice acceptor sites converted to the blunt-end restriction endonuclease sites Pml I and Pvu II, respectively. The blunt-end restriction endonuclease sites enabled the portable intron to be removed from the flanking adenovirus exon sequences and inserted into any blunt-end restriction endonuclease site in the recipient gene. After splicing, no adenovirus exon sequences remained in the recipient gene's RNA transcript. To demonstrate its usefulness, an insertion cassette was made by cloning the E. coli LacZ gene into a multiple cloning site within the type B intron. The insertion cassette was then cloned into a Pvu II site in the middle of the CAT gene. Following transfection in CEFs, high levels of both CAT and beta-Gal were detected, demonstrating that both genes were properly transcribed and translated.
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PMID:Construction of portable intron cassettes for the delivery and expression of foreign genes. 898 25

To study the involvement of DNA topoisomerase (topo) II on nonhomologous (illegitimate) recombination, we examined the effect of topo II inhibitors on random integration of exogenous vectors into human chromosomes. We transfected human cell lines PA1, HeLa and EJ-1 with linearized plasmid pSV2neo by electroporation, treated with topo II inhibitors and determined the frequency of Geneticin-resistant (G418r) colonies. We found that three topo II inhibitors, etoposide (VP-16), ICRF-193 and amsacrine (m-AMSA), greatly enhanced the frequency of G418r colonies. These effects were maximally expressed by as little as 12 hrs treatment with the drugs. Similar enhancements were found with different vectors (closed-circular and linear), different cell types, or by different transfection methods (calcium precipitation and lipofection). In contrast, the inhibitor treatments did not affect the transient expression of chloramphenicol acetyltransferase and beta-galactosidase activity following transfection with pSV2CAT and pCH110, respectively. Southern blot analysis revealed that the integration pattern of transfected pSV2neo into PA1 chromosomes was random and not characteristic for each inhibitor. These results suggest that topo II inhibitors directly act at a nonhomologous recombination reaction, promoting the integration process of transfected vectors into human chromosomes. We discuss the enhancement mechanism with a special emphasis on DNA strand breaks induced by the inhibitors.
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PMID:DNA topoisomerase II inhibitors enhance random integration of transfected vectors into human chromosomes. 900 Jan 72


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