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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)

Class II genes of the MHC show a striking homology upstream of the transcription start site that is composed of three conserved sequences (S, X and Y boxes, each separated by 15-20 bp). The presence of the S-box sequence in the mouse MHC class II gene I-A Beta was examined for its influence on the expression of this gene. Deletion or mutation of the S box decreased the induction of chloramphenicol acetyltransferase (CAT) activity in B lymphocytes by 32%. In macrophages, deletion or mutation of the S box abolished interferon-gamma (IFN-gamma) inducibility of CAT activity. Using a gel-retardation assay, we have identified a nuclear factor whose binding site overlaps the 7-mer conserved sequence of the S box. This factor is present in lymphocytes, macrophages, mastocytes and fibroblasts. Surprisingly, binding of this nuclear factor to DNA was induced by IFN-gamma in bone-marrow-derived macrophages, but not in macrophage-like cell lines. The binding site for this factor was defined by DNase I footprinting and partially purified by using an affinity column containing double-stranded oligonucleotides containing a sequence of the S box. A prominent protein of 43 kDa was found that bound specifically to the S-box sequence.
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PMID:Identification of a transcription factor that binds to the S box of the I-A beta gene of the major histocompatibility complex. 861 Nov 49

To investigate the mechanisms of transcriptional activation of interleukin-1beta (IL-1beta) in non-monocytic cells, we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL-1beta promoter and performed transient transfection experiments. We identified a promoter segment that activates transcription most efficiently in keratinocytes. Electrophoretic mobility shift assays (EMSA) with a 43-mer oligonucleotide derived from the functionally identified cis-acting element revealed specific complexes. By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift, transcription factor SP-1 or a closely related protein was shown to bind to this regulatory element. The closest match to the known SP-1 consensus sequence within the respective region is a TCCCCTCCCCT motif. Mutation of this motif almost completely, and specifically, abolished the binding of two low-mobility complexes and led to a 95% decrease of constitutive transcriptional activation of a reporter construct IL-1beta (-170/+108). Likewise, activation of this reporter construct by tumor necrosis factor-alpha depended on the SP-1 site. These observations suggest that a so-far-unrecognized SP-1 site in the human IL-1beta promoter may participate in the transcriptional regulation of this gene in keratinocytes.
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PMID:A novel SP-1 site in the human interleukin-1 beta promoter confers preferential transcriptional activity in keratinocytes. 897 97

O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer+) and -deficient (Mer-) cells, correlated with the endogenous MGMT activity in Mer+ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5'-CTGGGTCGC-3') for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer+ cells tested but was apparently restricted to the cytoplasm of Mer- cells. We conclude that the MEBP-enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer+ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer- cells.
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PMID:Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells. 911 92

A single-stranded 27-mer phosphorothioate oligodeoxynucleotide (ssPT) containing the transforming growth factor-beta (TGF-beta) response element was synthesized. Rat fetal lung fibroblasts were stably transfected with the ColCat 3.6 plasmid, which contains a portion of the 5'-flanking region of the proalpha1(I) collagen gene linked to the chloramphenicol acetyltransferase (CAT) gene. The cells were transiently transfected with the modified oligodeoxynucleotides in both the presence and absence of bleomycin, a fibrogenic antineoplastic agent. At 50 microg ssPT, the bleomycin-induced increase in CAT activity was abrogated. The ability of ssPT to inhibit collagen synthesis in rat fetal lung fibroblasts was determined. Single-stranded PTs inhibited both collagen synthesis and noncollagen protein synthesis induced by TGF-beta1, the mediator of the bleomycin fibrogenic effect. Inflamed granulation tissue fibroblasts were prepared from polyvinyl alcohol sponges implanted in the backs of rats. These fibroblasts were treated with various doses of ssPTs in the presence and absence of TGF-beta1. Single-stranded PTs also blocked both the TGF-beta1-induced increase in collagen synthesis and noncollagen synthesis in these fibroblasts. However, the TGF-beta1-induced increase in collagen and noncollagen protein synthesis was not blocked by ssPTs containing a mutated TGF-beta response element. In addition, ssPT did not significantly alter the basal levels of collagen and noncollagen protein synthesis in rat lung fibroblasts or in granuloma derived fibroblasts. Since dexamethasone was also able to block the TGF-beta1-induced increase in collagen and noncollagen protein synthesis (Meisler et al., [1997] J. Invest. Dermatol. 108:285-289), these data indicate that phosphorothioate oligodeoxynucleotide antifibrotic agents mimic the inhibitory effect of glucocorticoids on collagen synthesis without the untoward side effects of these steroids.
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PMID:Promoter competitors as novel antifibrotics that inhibit transforming growth factor-beta induction of collagen and noncollagen protein synthesis in fibroblasts. 1050 92

Exogenous triplex-forming oligodeoxynucleotides (TFO) have the capacity to modulate in vivo the expression of individual genes. As the administration of TFO to cells is not without problems, we analyzed the possibility of generating them directly in the cell, using specific expression vectors. We constructed three vectors, mU6-GA, mU6-CA, and mU6-CT, that direct the synthesis in human 293 cells of 76-mer CU, GU, and AG motif TFO (rTFO) potentially capable of binding to a critical poly (R x Y) sequence contained in the promoter of the Ki-ras proto-oncogene. The ability of the CU, GU, and AG motif rTFO to interact with the double helix of the c-Ki-ras target was investigated in vitro by footprinting and band-shift experiments, using both synthetic and endogenously synthesized oligoribonucleotides. The human 293 cells were transfected with DNA mixtures containing a plasmid, which bears the reporter chloramphenicol acetyltransferase (CAT) gene downstream from the c-Ki-ras promoter (pKRS-413), as well as an rTFO-generating vector (mU6-GA, mU6-CA, or mU6-CT). As control, the cells were transfected with DNA mixtures containing vector mU6-C1 or mU6-C2. These generated transcripts unable to form triple helices with the poly (R x Y) sequence of the c-Ki-ras promoter. Intracellular synthesis of the 76-mer CU, GU, and AG rTFO by mU6-GA, mU6-CA, and mU6-CT was checked by Northern blot hybridization. Through beta-gal and CAT ELISA immunoassays, we found that the 293 cells transfected with either mU6-GA, mU6-CA, or mU6-CT showed a significant inhibition of CAT expression compared with cells transfected with control plasmids mU6-C1 or mU6-C2. The results of five separate transient transfection experiments showed that endogenous GU and AG rTFO, generated by mU6-CA and mU6-CT, produce, respectively, 40% (+/- 4% SE) and 47% (+/- 8% SE) CAT inhibition, whereas CU rTFO, generated by mU6-GA, produces 38% (+/- 7% SE) CAT inhibition. In conclusion, this study suggests that it is possible to downregulate the expression of an individual gene through the use of recombinant vectors encoding the information for the intracellular synthesis of short triplex-forming RNA strands.
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PMID:Downregulation of c-Ki-ras promoter activity by triplex-forming oligonucleotides endogenously generated in human 293 cells. 1098 22

The murine Ki-ras promoter contains a unique polypurine--polypyrimidine [poly(R.Y)] sequence between -290 and -320 from the 3' boundary of exon phi. Previously we demonstrated triplex formation and transcription inhibition promoted by GT and AG oligonucleotides directed against this site [Alunni-Fabbroni et al. (1996) Biochemistry 35, 16361--16369]. In this work, we have investigated triplex formation and anti-gene activity of five 20-mer AG motif triplex-forming oligonucleotides specific for the Ki-ras poly(R.Y) target, derived from 5'-AGGGAGGGAGGAAGGGAGGG (20AG) by replacing an increasing number of phosphodiester linkages with phosphorothioate linkages (S(i)-20AG; i = 2, 3, 4, 5, 19). Electrophoretic mobility-shift experiments (EMSA) showed that four thioate oligonucleotides, S(i)-20AG (i = 2, 3, 4, 5), recognized the Ki-ras target and exhibited dissociation constants similar to that of 20AG: K(d) = 12 +/- 2 nM, while the all-thioate S(19)-20AG exhibited a K(d) of 128 +/- 15 nM. Moreover, the binding between the Ki-ras promoter and oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) was characterized by DMS/piperidine and DNase I footprinting experiments. We observed that the introduction in the phosphodiester oligonucleotide 20AG of sulfur atoms reduced its aggregation significantly and increased its nuclease resistance. Transient transfection experiments using preformed triplexes with a recombinant plasmid containing the reporter chloramphenicol acetyltransferase (CAT) gene under the control of Ki-ras promoter showed that oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) promote a strong inhibition of up to 75% of the CAT expression when compared with control Ki-ras unspecific oligonucleotides. Taken together, these data provide a guideline for designing triplex-forming effector molecules capable of controlling Ki-ras expression in vivo.
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PMID:Anti-gene effect in live cells of AG motif triplex-forming oligonucleotides containing an increasing number of phosphorothioate linkages. 1117 Apr 38

We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the Nano-tag(9) is a 9-mer peptide with a dissociation constant of 17 nM. We demonstrate the one-step purification of Nano-tagged proteins, namely bovine heart fatty acid-binding protein (FABP), bacterial chloramphenicol acetyltransferase (CAT), and green fluorescent protein (GFP), from an in vitro translation system as well as from an Escherichia coli lysate. No significant influence of the Nano-tag(15) and of the conditions during affinity chromatography on maturation or activity of the proteins was observed whereas the Nano-tag(9) revealed a slight decline in the amount and activity of the synthesized proteins. The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state. Additionally, the Nano-tag allowed the detection of recombinant proteins on Western blots by a streptavidin-alkaline phosphatase conjugate.
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PMID:The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins. 1468 Sep 60


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