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)

Sonicated liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and a quaternary ammonium detergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide) mediates functional transfer of pSV2 CAT plasmid DNA to mouse L929 fibroblasts. Successful transfection was determined by assaying for chloramphenicol acetyltransferase activity in cell lysates collected 40 h after exposure to the lipid-DNA complexes. Liposomes prepared with the quaternary ammonium detergents were less toxic than the free detergents at the same concentrations and were more efficient in their delivery of the plasmid DNA to the cells. Analysis of the three detergents in combination with the lipid showed that cetyltrimethylammonium bromide was least toxic to the cells. This detergent, at a minimal concentration of 20 mol% in DOPE, allowed for stable liposome preparations and efficient transfection. Optimal efficiency of transfection occurred with 30 micrograms of DNA. Further increases in the DNA concentration caused a decrease in the transfection efficiency, perhaps due to charge repulsions between the liposomes now saturated with negatively charged DNA and the negatively charged cell surface. The transfection activity of the liposome was limited by its cytotoxicity at high liposome concentrations. These results are compared with that of the Lipofectin, another positively charged liposome preparation which is commercially available. Although the overall transfection activity of the liposome containing the quaternary ammonium detergent is somewhat lower than that of the Lipofectin, it may serve as an inexpensive and convenient alternative.
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PMID:Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells. 279 44

It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.
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PMID:Cell poration and cell fusion using an oscillating electric field. 281 30

Expression of the prokaryotic gene for chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT) in primate cells transfected with X-irradiated plasmid pSV2CAT was determined in transient expression assays. CAT expression did not depend upon the presence of supercoiled plasmids, but relaxed circular forms were essential. X-ray conversion of relaxed circles to linear forms paralleled the loss of CAT expression, with identical D0's in the first part of dose-response curves. X-ray-induced loss of supercoiled forms was complete at much lower doses. The D0 for inactivation of CAT expression by X irradiation of the plasmids in 1 mM Tris buffer was 270 Gy; it was 13 Gy for plasmids irradiated in water. The D0's for conversion of pSV2CAT to relaxed circle forms were only one-seventh as large as the D0's for CAT inactivation after X-ray in water or in 1 mM Tris buffer. Expression of the CAT gene in some representative repair-deficient human fibroblasts transfected with X-irradiated pSV2CAT was less than in monkey CV-1 cells or cell lines from normal human subjects. These results demonstrate a novel means to study low levels of X-ray damage in DNA correlating specific X-ray damage in the DNA with expression of the gene in unirradiated primate cells.
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PMID:Expression of prokaryotic genes after transfection of X-irradiated plasmids into primate cells. 281 35

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

The late E2A promoter of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promoter. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W. Doerfler, Proc. Natl. Acad. Sci. USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci. USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promoter-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promoter methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promoter controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promoter controls the gene for neomycin phosphotransferase. The pAd2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuration. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAd2E2AL-CAT construct, the late E2A promoter remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promoter remained almost completely methylated. In five cell lines, the E2A promoter sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylations were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promoter, after this promoter was fixed by integration in the mammalian genome.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promoter-cat gene construct in the genome of hamster cells: gene expression and stability of methylation patterns. 282 9

We used the Escherichia coli chloramphenicol acetyltransferase gene (cat) to study sequences that influence expression of the equine infectious anemia virus (EIAV) genome. The EIAV long terminal repeat (LTR) directed CAT activity in a canine cell line, but at levels much lower than those achieved with other eucaryotic viral promoters. In the same cells infected with EIAV or cotransfected with molecularly cloned EIAV genomic DNA, LTR-directed activity was markedly enhanced. Comparison of cat mRNA and protein levels in these cells indicated that this trans-activating effect could be accounted for by a bimodal mechanism in which both transcriptional and posttranscriptional events are enhanced. trans-Activation but not promoter activity was abolished by deletion of the R-U5 region of the EIAV LTR. EIAV sequences responsible for the trans-activating function could be localized to a region encompassing the 3' and 5' termini of the pol and env genes, respectively (nucleotides 4474 to 5775). Interestingly, this stretch harbors a short open reading frame with some amino acid sequence similarity to the human immunodeficiency virus type I tat gene product.
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PMID:Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat. 282 40

Almost all homosexual patients with acquired immunodeficiency syndrome are also actively infected with human cytomegalovirus (HCMV). We have hypothesized that an interaction between HCMV and human immunodeficiency virus (HIV), the agent that causes acquired immunodeficiency syndrome, may exist at a molecular level and contribute to the manifestations of HIV infection. In this report, we demonstrate that the immediate-early gene region of HCMV, in particular immediate-early region 2, trans-activates the expression of the bacterial gene chloramphenicol acetyltransferase that is fused to the HIV long terminal repeat and carried by plasmid pHIV-CAT. The HCMV immediate-early trans-activator increases the level of mRNA from the plasmid pHIV-CAT. The sequences of HIV that are responsive to trans-activation by the HCMV immediate-early region are distinct from HIV sequences that required for response to the HIV tat. The stimulation of HIV gene expression by HCMV gene functions could enhance the consequences of HIV infection in persons with previous or concurrent HCMV infection.
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PMID:Immediate-early gene region of human cytomegalovirus trans-activates the promoter of human immunodeficiency virus. 282 1

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [14C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.
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PMID:A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals. 282 96

Transfection into myogenic and nonmyogenic cell lines was used to investigate the transcriptional regulation of the human alpha-skeletal actin gene. We demonstrated that 1,300 base pairs of the 5'-flanking region directed high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated mouse C2C12 and rat L8 myotubes but not in mouse nonmuscle L.TK- and HuT-12 cells. Unidirectional 5' deletion analysis and heterologous promoter stimulation experiments demonstrated that at least three transcription-regulating subdomains lie in this 1,300-base-pair region. A proximal cis-acting transcriptional element located between positions -153 and -87 relative to the start of transcription at +1 was both sufficient and necessary for muscle-specific expression and developmental regulation during myogenesis in the two myogenic cell systems. The region 3' of position -87 interacted with factors present in both myogenic and fibroblastic cells and appeared to define, or to be a major component of, the basal promoter. In C2C12 myotubes, but not in L8 myotubes, a distal sequence domain between positions -1300 and -626 and the proximal sequence domain between positions -153 and -87 each induced transcription about 10-fold and synergistically increased CAT expression 100-fold over levels achieved by the sequences 3' of position -87. Furthermore, these cis-acting elements independently and synergistically modulated an enhancerless, heterologous simian virus 40 promoter in a tissue-specific manner. DNA fragments which included the proximal domain displayed classical enhancerlike properties. The central region between positions -626 and -153, although required in neither cell line, had a positive, two- to threefold, additive role in augmenting expression in L8 cells but not in C2C12 cells. This suggests that certain elements between positions -1300 and -153 appear to be differentially utilized for maximal expression in different myogenic cells and that the particular combination of domains used is dependent on the availability, in kind or amount, of trans-acting, transcription-modulating factors present in each cell type. Thus, multiple myogenic factors that vary qualitatively and quantitatively may be responsible for the different and complex modulatory programs of actin gene expression observed during in vivo muscle differentiation.
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PMID:Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression. 282 26

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


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