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)

Hepatocyte growth factor (HGF) is a multifunctional cytokine that controls the growth and differentiation of various tissues. Previously, we described the existence of a negative cis-acting regulatory element(s) within the -1- to -0.7-kilobase pair (kb) portion of the 5'-flanking region of the mouse HGF promoter. In the present study, we show that the repressor element is located at position -872 to -860 base pairs and comprises an imperfect estrogen-responsive element 5'-AGGTCAGAAAGACCA-3'. We demonstrate that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a nuclear orphan receptor belonging to the steroid/thyroid hormone receptor superfamily, through binding to this site effectively silences the transcriptional activity of the HGF promoter. We show that estrogen receptor, on the other hand, relieves the repressive action of COUP-TF, resulting in the induction of the HGF promoter. Using mice transgenic for either 2.7 or 0.7 kb of the HGF promoter region linked to the chloramphenicol acetyltransferase reporter gene, we found that injection of estradiol stimulates HGF promoter activity in tissues such as the mammary gland and ovary of mice harboring 2.7 but not 0.7 kb of the mouse HGF promoter region. Potential involvement of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors in the regulation of HGF gene expression is also discussed.
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PMID:Transcriptional regulation of the hepatocyte growth factor gene by the nuclear receptors chicken ovalbumin upstream promoter transcription factor and estrogen receptor. 902 96

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from -1645 to -1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region a fragment from -1645 to -1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from -1605 to -1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] -1635 to -1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt -1599 to -1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.
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PMID:Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells. 903 89

Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-specific genes in heterologous cell types, such as embryonic fibroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, especially the human homolog of chicken NF-M, C/EBP-beta (NF-IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inflammatory stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of primers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel human gene encoding a member of the C/EBP gene family, identified as the human homolog of CRP1, C/EBP-epsilon. A 1.2-kb cDNA encoding full-length human C/EBP-epsilon was cloned from a promyelocyte-late myeloblast-derived lambda gt11 library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer extension analysis of C/EBP-epsilon mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-specific genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the factors of the Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipitation studies using rabbit anti-C/EBP-epsilon antibodies raised against the N-terminal portion of C/EBP-epsilon (amino acids 1 to 115) showed that C/EBP-epsilon is a 32-kDa nuclear phosphoprotein. The human C/EBP-epsilon protein exhibited strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of the C/EBP-epsilon sense and antisense expression constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-specific c-mim and human myeloperoxidase promoters suggested a role for C/EBP-epsilon transcription factor in the regulation of a subset of myeloid-cell-specific genes. Transient tranfection of a promyelocyte cell line (NB4) with a C/EBP-epsilon expression plasmid increased cell growth by sevenfold, while antisense C/EBP-epsilon caused a fivefold decrease in clonal growth of these cells.
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PMID:Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor. 903 64

C-terminally truncated surface proteins of hepatitis B virus (HBV) are frequently translated from genomically integrated viral sequences. They may be relevant for hepatocarcinogenesis by stimulating gene expression. First, we examined the transactivating potential of middle hepatitis B surface protein truncated at amino acid (aa) position 167 (MHBst167) on the HBV regulatory element. In transient cotransfection assays using Chang liver or HepG2 cell lines and chloramphenicol acetyltransferase (CAT) reporter constructs only the HBV enhancer I, but no other HBV regulatory elements like the X promoter, the S1 or S2 promoter or the enhancer II/core promoter could be stimulated by MHBst167. Since there is no evidence for a direct interaction of MHBst167 with DNA, we subsequently analysed whether cellular transcription factors were involved in mediating transactivation. This was tested both with isolated transcription-factor-binding sites and in the natural context of viral and cellular promoter elements. Deletion analysis and electrophoretic mobility shift assays revealed that Sp1, AP1 and NF-kappa B can mediate transactivation by MHBst167. No involvement of CREB, NF1 or the liver-specific factor C/EBP was found. These data indicate that MHBst167 is a pleiotropic, non-liver-specific transactivator which exerts its effect via ubiquitous cellular transcription factors that are also involved in the regulation of expression of cellular genes relevant for proliferation and inflammation.
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PMID:The hepatitis B virus MHBst167 protein is a pleiotropic transactivator mediating its effect via ubiquitous cellular transcription factors. 919 47

Paclitaxel (Taxol) is a novel chemotherapeutic drug that is effective against breast and ovarian cancers. Although the primary target of paclitaxel is microtubules, its efficacy exceeds that of conventional microtubule-disrupting agents, suggesting that it may have additional cellular effects. Previously, we demonstrated that paclitaxel can induce interleukin-8 (IL-8) gene expression at the transcriptional level in subsets of human ovarian cancer lines. In this as well as the previous report, we present evidence that this ability is not linked to the lipopolysaccharide pathway of IL-8 gene induction. The present study identifies the cis-acting elements and trans-acting factors involved in this induction by transfecting DNA constructs containing the 5'-flanking region of the IL-8 gene linked to the chloramphenicol acetyltransferase reporter gene into paclitaxel-responsive and nonresponsive ovarian cancer cells (responsiveness refers to the IL-8 response). Paclitaxel only activated the IL-8 promoter in responsive cells. The AP-1 and NF-kappaB binding sites in the IL-8 promoter are required for activation by paclitaxel; in contrast, a C/EBP site required for IL-8 promoter activation in other cell types is not involved. Gel shift assays demonstrate that paclitaxel causes a marked increase in protein binding to the NF-kappaB and AP-1 consensus binding sequences in the paclitaxel-responsive ovarian cells, but not the nonresponsive cells. The induction of NF-kappaB and AP-1 binding is reduced by the addition of protein kinase C inhibitors and cyclic AMP effector, respectively. These results demonstrate a molecular mechanism for cell-specific paclitaxel-induced IL-8 gene expression which may have clinical relevance.
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PMID:Identification of tumor-specific paclitaxel (Taxol)-responsive regulatory elements in the interleukin-8 promoter. 927 87

Recently, we demonstrated that the function of ATF3, a stress-inducible transcriptional repressor, is negatively regulated by a bZip protein, gadd153/Chop10. In this report, we present evidence that ATF3 can repress the expression of its own inhibitor, gadd153/Chop10. First, ATF3 represses a chloramphenicol acetyltransferase reporter gene driven by the gadd153/Chop10 promoter when assayed by a transfection assay in vivo and a transcription assay in vitro. Second, the gadd153/Chop10 promoter contains two functionally important binding sites for ATF3: an AP-1 site and a C/EBP-ATF composite site, a previously unidentified binding site for ATF3. The absence of either site reduces the ability of ATF3 to repress the promoter. Third, overexpression of ATF3 by transient transfection results in a reduction of the endogenous gadd153/Chop10 mRNA level. Fourth, as described previously, ATF3 is induced in the liver upon CCl4 treatment. Intriguingly, we show in this report that gadd153/Chop10 mRNA is not present in areas where ATF3 is induced. Taken together, these results strongly suggest that ATF3 represses the expression of gadd153/Chop10. The mutual negative regulation between ATF3 and gadd153/Chop10 is discussed.
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PMID:gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3. 934 34

To identify the 5' sequences of the murine coagulation factor VII (fVII) gene that resulted in its efficient transcription, a variety of 5'-flanking sequences up to 7 kilobase pairs upstream of the translation ATG initiation codon were fused to the reporter gene, bacterial chloramphenicol acetyltransferase, and relative expression levels of this gene in mouse Hepa 1-6 cells were determined. It was found that the 5' region extending approximately 85 base pairs (bp) upstream of the transcriptional initiation site served as the minimal DNA region that provided full relative promoter activity for chloramphenicol acetyltransferase expression. This region of the gene also contains consensus sequences for liver-enriched transcription factors, C/EBP beta and HNF4, as well as for the ubiquitous protein factors, AP1, H4TF1, NF1, and Sp1. In vitro DNase I footprinting of the 200-bp proximal region of the promoter with a murine Hepa 1-6 cell nuclear extract revealed a clear footprint of a region corresponding to -80 to -28 bp of the murine fVII gene, suggesting that liver factors interact with this region of the DNA. Competitive gel shift and supershift assays with different synthetic oligonucleotide probes demonstrate that proteins contained in the nuclear extract, identified as C/EBP beta, H4TF1, and HNF4, bind to a region of the murine fVII DNA from 85 to 32 bp upstream of the transcription start site. Purified Sp1 also interacts with this region of the DNA at a site that substantially overlaps, but is not identical to, the H4TF1 binding locus. Binding of Sp1 to the mouse DNA was not observed with the nuclear extract as the source of the transcription factors, suggesting that Sp1 is likely displaced from its binding site by H4TF1 in the crude extract. In vivo dimethyl sulfate footprint analysis confirmed the existence of these sites and additionally revealed two other binding regions slightly upstream of the CCAAT/enhancer-binding protein (C/EBP) binding locus that are homologous to NF1 binding sequences. The data demonstrate that appropriate transcription factor binding sites exist in the proximal promoter region of the murine fVII gene that are consistent with its strong liver-based expression in a highly regulated manner.
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PMID:Characterization of transcriptional regulatory elements in the promoter region of the murine blood coagulation factor VII gene. 944 72

We present here the cloning and characterization of the EhPgp1 multidrug resistance gene promoter isolated from the Entamoeba histolytica drug-resistant mutant clone C2. The EhPgp1 promoter lacks the typical TATA box and the transcriptional initiation sequences described for other E. histolytica promoters. The major transcription initiation site of the EhPgp1 gene was located at the ATG start codon. The EhPgp1 core promoter located within the first 244 base pairs showed a higher chloramphenicol acetyltransferase expression in the transfected trophozoites of clone C2 than in those of the sensitive clone A. Gel shift assays revealed three specific DNA-protein complexes (Ia, IIa, and IIIc) using nuclear extracts from clone C2, whereas three main complexes (If, IIf, and IIg) were limited to clone A. Competition assays suggested the presence of C/EBP-like and OCT-like proteins in complexes Ia and IIa, respectively, probably involved in the expression of the EhPgp1 gene, whereas complex IIIc was competed by GATA-1, C/EBP, OCT, and HOX oligonucleotides. Thus, differential DNA-protein complexes may be formed by transcriptional factors involved in the regulation of the EhPgp1 gene expression.
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PMID:Transcriptional analysis of the EhPgp1 promoter of Entamoeba histolytica multidrug-resistant mutant. 951 21

Expression of the inducible nitric oxide synthase (iNOS) gene in rat mesangial cells is differentially triggered by IL-1beta and cAMP predominantly at the transcriptional level. The 5'-flanking region of the rat iNOS gene contains several binding sites for transcription factors potentially involved in cytokine and cAMP signaling such as nuclear factor-kappaB/Rel, CCAAT/enhancer-binding protein (C/EBP), and cyclic AMP-responsive element-binding protein/ATF. We tested promoter activities of serial and site-directed deletion mutants of iNOS-chloramphenicol acetyltransferase reporter genes after transient transfection and stimulation of mesangial cells. A region between bp -277 and -111 bearing a CCAAT/enhancer-binding protein-response element was found to be critical for cAMP-mediated gene induction but dispensable for IL-1beta inducibility. Moreover, a minimal promoter ranging from the transcriptional start site up to -111 containing a kappaB site is sufficient to confer IL-1beta-mediated iNOS promoter activation. Consistent with these findings, an electrophoretic mobility shift assay shows the appearance of an IL-1beta-inducible nuclear factor-kappaB p50/p65 heterodimeric complex. Using probes containing C/EBP-binding sites from the iNOS gene revealed further binding of different complexes, all of which were strongly inducible by cAMP and to a lower extent also by IL-1beta. Abs against cyclic AMP-responsive element-binding protein, C/EBPbeta, and C/EBPdelta were able to partially supershift single complexes, suggesting the participation of these transcription factors in the regulation of iNOS gene expression by cAMP and IL-1beta. Finally, we show that both cAMP and IL-1beta strongly induce steady-state levels of C/EBPbeta and C/EBPdelta mRNA levels. These data demonstrate that IL-1beta and cAMP use distinct as well as partially overlapping sets of transcriptional activators to modulate iNOS gene expression in rat mesangial cells.
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PMID:Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells. 959 Feb 44

Human CCAAT/enhancer-binding protein epsilon (C/EBPepsilon), a new member of the C/EBP family, significantly up-regulates both the mim-1 and human myeloperoxidase promoters, suggesting an important role for C/EBPepsilon in the transcriptional regulation of a subset of myeloid-specific genes. To elucidate the structure and function of C/EBPepsilon in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBPepsilon were fused to the yeast GAL4 DNA binding domain. These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the GAL-C/EBPepsilon-(1-115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene. Sixteen deletion mutants of C/EBPepsilon mapped the transactivation domain to amino acids 1-18 at the N terminus and revealed the presence of a transcription repression element between amino acid residues 116 and 162. Expression vectors containing the repression domain of C/EBPepsilon strongly inhibited gene transcription from TK, SV40, and adenoviral major late promoters bearing GAL4 binding sites. Fusion of this repression domain to the VP16 activation domain inhibited the transactivation function of VP16. Deletion of this repression domain increased gene transcription from a neutrophil elastase promoter-luciferase reporter. Taken together, these data suggest that C/EBPepsilon regulates transcription by utilizing both activation and repression functions.
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PMID:Identification of transcriptional activation and repression domains in human CCAAT/enhancer-binding protein epsilon. 961 80


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