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)

Human foamy virus (HFV) encodes the transcriptional transactivator bel1. The bel1 protein transactivates HFV long terminal repeat (LTR)-directed gene expression by recognizing a region in U3. It also transactivates human immunodeficiency virus type 1 (HIV-1) LTR-directed gene expression in transient transfection assays. To identify the specific region in HIV-1 LTR responsible for bel1 action, we examined the effect of bel1 on chloramphenicol acetyltransferase (CAT) gene expression in transfected cells with a series of mutant HIV-1 LTR/CAT plasmids. The region between -158 and -118 from the transcription initiation site, immediately upstream of the core enhancer element, was identified as responsible for the transactivation by bel1. In addition, bel1 transactivated a heterologous promoter when this region was positioned upstream of it in the sense and antisense orientations. Optimal transactivation of the HIV-1 LTR by bel1 did not require an intact TAR sequence, suggesting that the binding of tat to the TAR sequence is not a prerequisite for bel1 function in HIV-1 LTR-directed gene expression. In the region of the HIV-1 LTR that is necessary for the bel1-mediated transactivation, we have found a sequence which is conserved between HIV-1 and HFV. Our results suggest that the bel1 action on HIV-1 seems to be mediated by a specific DNA sequence which is shared by both the HIV-1 LTR and HFV LTR.
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PMID:Transactivation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by the human foamy virus bel1 protein requires a specific DNA sequence. 131 28

Angiotensinogen is a precursor of the multifunctional octapeptide hormone, angiotensin II. We have isolated the overlapping clones containing angiotensinogen gene locus from C57BL/6 mouse genomic DNA library and analyzed them by restriction enzyme mapping. The gene exhibited a structural organization similar to those of the human, rat and balb/c mouse angiotensinogen genes. Using a genomic DNA fragment of the mouse angiotensinogen gene as a probe, we have investigated the tissue distribution of angiotensinogen messenger RNA (mRNA) in C57BL/6 mouse. The angiotensinogen mRNA was highest in the liver and detectable in such tissues as brain, kidney, submandibular gland, ovary and heart. However, it was undetectable in lung and spleen under the condition used. Optimal alignments of the 5'-flanking regions among the human, rat and mouse angiotensinogen genes disclosed several deletions in the mouse sequence. To assay the promoter activity, the 5'-flanking region of the mouse angiotensinogen gene was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene, then transfected into different cultured cells. The angiotensinogen gene sequences elicited preferential expression of CAT activity when introduced into HepG2 cells derived from liver and 293 cells from kidney but not in HeLa cells from uterus, suggesting the presence of a cell type-specific promoter within the sequences. These findings on the structure and expression of the mouse angiotensinogen gene should prove useful in studying the function and control of the angiotensin.
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PMID:Structure and expression of the mouse angiotensinogen gene. 157 74

The ability of the promotor/enhancer region of the mouse ornithine decarboxylase gene to respond to various stimuli was studied. This region was subcloned into multiple fragments and these were inserted in front of the chloramphenicol acetyltransferase gene on an expression vector, pBLCAT3. These ODC/CAT constructs were transfected into a mouse macrophage-like cell line, RAW264. The transfected cells were stimulated by bacterial lipopolysaccharide, 8-bromo cAMP or both followed by analysis of chloramphenicol acetyltransferase activity. Optimal inducible chloramphenicol acetyltransferase expression was obtained when sequences from -90 to +12 (with respect to the transcriptional start site) were tested in cells treated with a combination of lipopolysaccharide and 8-bromo cAMP. A putative cyclic AMP response element located at -48 was altered by site-directed mutagenesis but these alterations did not diminish activity in response to stimulation with lipopolysaccharide and 8-bromo cAMP.
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PMID:Regulation of mouse ornithine decarboxylase gene expression in a macrophage-like cell line: synergistic induction by bacterial lipopolysaccharide and cAMP. 184 9

The transcription rate of the haptoglobin (Hp) gene is stimulated by interleukin-1 (IL-1), IL-6, and dexamethasone in rat hepatoma (H-35) cells. To identify the cis-acting regulatory elements responsive to these hormones, various lengths of 5' Hp gene-flanking regions, including the promoter, were inserted into chloramphenicol acetyltransferase gene expression vectors and transiently introduced into H-35 cells. The first 4 kb of 5' region mediated a severalfold increase in expression after treatment with IL-6 and dexamethasone. No response to IL-1 was detectable. When, however, upstream sequences were deleted to position -165 relative to the transcription start site, a significant stimulation by IL-1 was gained without appreciably affecting the IL-6 response. With the apparent removal of an inhibitory sequence, the promoter-proximal 165-bp region also displayed a severalfold enhanced response to the combination of dexamethasone, IL-1, and IL-6. The sequence from -165 to -147, termed the A-element, was found to be crucial for all hormone regulatory functions. Two copies of the A-element linked to a heterologous promoter responded to the three hormones, but to a lesser degree than in the Hp gene promoter context. The regulatory elements of the rat Hp gene were similarly active in human hepatoma cells. Optimal regulation by IL-6 in HepG2 cells was, however, independent of the A-element. The A-element functioned in these cells exclusively as an IL-1 response sequence. The results suggest that genomic sequences upstream of the rat Hp gene suppress the regulation by specific cytokines more prominently in transient expression assays than in the normal chromosomal context. Moreover, the functional comparison indicated that specific regulatory regions of the rat Hp gene do not function identically in different hepatic cell types.
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PMID:Distinct regulation of the interleukin-1 and interleukin-6 response elements of the rat haptoglobin gene in rat and human hepatoma cells. 217 89

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

A simple method for introducing genes into respiratory epithelial cells would assist molecular studies of a variety of pulmonary disorders. Several different techniques for introducing foreign DNA into cells have been described but have either not been useful for respiratory epithelial cells or are difficult and cumbersome to perform. Electroporation is a simple technique that consists of exposing a cell-DNA suspension to an electric shock. Although it has been used to introduce genes into a variety of cell types, it has not previously been applied to respiratory epithelial cells. Human nasal epithelial cells were transfected with the plasmid pRSVCAT, which is an expression vector containing the origin of replication of pBR322 coupled to the Rous sarcoma virus (RSV) long terminal repeat (LTR) region driving the coding sequence for the chloramphenicol acetyltransferase (CAT) gene. The CAT gene is useful for determining optimal conditions for electroporation since it is not normally present in eukaryotic cells, and CAT activity correlates with the level of CAT mRNA; this provides a measure of expression of introduced foreign genes. Successful expression of the CAT gene was demonstrated by electroporation, whereas calcium phosphate transfection resulted in very low CAT activity. Optimal conditions for electroporation of respiratory epithelial cells were determined. Electroporating nasal epithelial cells using 500 volts, a DNA concentration of 10 micrograms/ml, and a sucrose buffer yielded the highest CAT activity, which peaked at 48 h after electroporation.
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PMID:The introduction of biologically active foreign genes into human respiratory epithelial cells using electroporation. 284 46

We have isolated the human apolipoprotein (apo) A-IV gene from a cosmid library and determined its complete nucleotide sequence. The gene contains three exons of 162, 127, and 1180 nucleotides separated by two introns of 357 and 777 nucleotides. A sequence polymorphism has been identified in the 3' noncoding portion of the third exon. The human apoA-IV gene lacks an intron in the area encoding the 5' nontranslated region of its mRNA, which distinguishes it from all the other human apolipoprotein genes whose sequences are known. Comparison matrix analysis of the human apoA-IV gene sequence revealed evidence for an ancestral 11-nucleotide repeat unit that spans the third exon. These repeated sequences are much more highly conserved than those present in either rat apoA-IV or in any other human apolipoprotein. Optimal alignments of the 5' flanking regions of the rat and human apoA-IV genes disclosed multiple deletions in the rat sequence as well as a highly conserved region of 90 nucleotides (90% sequence identity) located within 170 nucleotides of the start site of transcription. The 5' flanking regions of the human and rat apoA-IV genes were ligated to the bacterial chloramphenicol acetyltransferase gene, then transfected into different cultured cells. The apoA-IV gene sequences elicited preferential expression of chloramphenicol acetyltransferase activity when introduced into intestinally derived Caco-2 cells and liver-derived Hep-G2 cells, consistent with the tissue specificity of the native gene. Analysis of deletion mutants of the human apoA-IV 5' flanking region indicated that regions from -293 to -233 and from -127 to -60 upstream of the transcription start site contain sequences required for maximum gene expression. These findings on the structure and expression of rat and human apoA-IV should prove useful in studying the control of the apoA-IV gene.
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PMID:Structure and expression of the human apolipoprotein A-IV gene. 303 93

The polycation 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) is superior to calcium phosphate for the introduction of purified DNA into cultured Aedes albopictus (mosquito) cells. Adsorption of the polybrene-DNA complex to mosquito cells was essentially linear for 6 h. However, the rate of adsorption of DNA increased when the DNA-polybrene mixture was preincubated for several hours prior to addition to cells. A recombinant plasmid carrying an inducible chloramphenicol acetyltransferase gene under the control of a Drosophila heat shock protein (hsp) promoter was used to show that expression of transfected DNA was highest when cells were treated with a freshly prepared polybrene-DNA mixture. Optimal expression was observed in cells transfected with 4-13 micrograms of DNA per 10(6) cells; transfection with 24 micrograms of DNA resulted in reduced CAT expression. Variation in the polybrene-DNA ratio improved transfection with high levels of DNA. In mosquito cells, CAT expression was independent of DNA methylation.
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PMID:Factors affecting polybrene-mediated transfection of cultured Aedes albopictus (mosquito) cells. 346 5

We have developed a system using explanted embryonic chicken lens epithelia to express foreign recombinant genes containing crystallin DNA regulatory sequences introduced by calcium phosphate transfection. Optimal results were obtained with lens epithelia from 14-day embryos transfected 1 day after explantation and assayed 3 days later. When DNA sequences (-364 to +45) of the murine alpha A-crystallin gene were inserted in the pSVO-CAT expression vector of Gorman et al. [Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051] in the same orientation as in the crystallin gene, they promoted chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) activity in the transfected epithelia. Sequences 87 to 364 base pairs upstream from the murine gene cap site were required for CAT gene expression. These crystallin gene regulatory sequences did not promote CAT expression in primary cultures of embryonic chicken fibroblasts or other nonlens cells. By contrast, the long terminal repeat of Rous sarcoma virus and the early promoter of simian virus 40 promoted CAT activity in lens and nonlens cells. Our experiments thus demonstrate that the explanted embryonic chicken lens epithelium is an advantageous recipient for identifying lens-cell-specific regulatory sequences of crystallin genes and implicate a DNA region upstream of the "TATA box" for regulation of the murine alpha A-crystallin gene. These experiments also suggest that explanted epithelia from other tissues may be useful for studying the expression of foreign genes.
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PMID:Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia. 385 84

Optimal activation of T cells requires at least two signals. One signal can be delivered by the antigen-specific T-cell receptor, and the second signal is provided by the costimulatory molecule(s) delivered by the antigen-presenting cell. CD28 is a T-cell surface molecule and stimulation through this protein plays an important role in delivering the second activation signal. In this report, we show that in human peripheral blood T cells, CD28-mediated signal transduction involves the rel family proteins--c-Rel, p50, and p65. Treatment of peripheral blood T cells with phorbol 12-myristate 13-acetate (PMA) and anti-CD28 monoclonal antibody (mAb) results in augmentation of nuclear c-Rel, p50, and p65, and this augmentation can occur in the presence of the immunosuppressant cyclosporin A. It is also shown in this report that, in response to PMA/anti-CD28 mAb or anti-CD3/anti-CD28 mAb, c-Rel, p50, and p65 are associated with CD28-responsive element present in the promoter of the human interleukin 2 gene. The functional significance of c-Rel involvement in the CD28-responsive complex is demonstrated by transient transfection analysis, where cotransfection of c-Rel augments the level of expression of a chloramphenicol acetyltransferase reporter gene linked to the CD28-responsive element.
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PMID:The interleukin 2 CD28-responsive complex contains at least three members of the NF kappa B family: c-Rel, p50, and p65. 838 23


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