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)

The 5'-flanking region of the human brain-derived neurotrophic factor (BDNF) gene was isolated from a human placental genomic library using the cDNA fragment for the 5'-noncoding region of human BDNF as a probe. A 3.2 Kbp genomic fragment containing the 5'-flanking region, the first exon and a portion of the first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 nuclease mapping, was located 26 bp downstream from the TATA-like sequence. Several expression plasmids, in which the BDNF promoter regions were fused to the chloramphenicol acetyltransferase (CAT) gene, were constructed. Transient expression in human glioma Hs683 cells demonstrated that a fragment of about 0.5 Kbp from the transcriptional initiation site was sufficient for promoter activity.
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PMID:Characterization of the 5'-flanking region of the human brain-derived neurotrophic factor gene. 133 67

We have recently isolated a functional promoter encoding the human polypeptide-binding protein (BiP) gene from Burkitt's lymphoma cells by polymerase chain reaction (The EMBL Data Library accession number X59969, 1991). This promoter DNA segment (termed BiP670) was fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and expressed in NIH3T3 cells. BiP670 retains basal and Ca2+ ionophore A23187-inducible activities. Using 5' deletion assay, we found three basal expression elements (BEE) in the BiP670. Removal of the distal BBE (BBE3), which is contained in a segment spanning -368/-170, caused a 50% loss of the basal activity; removal together with the middle BBE (BBE2), which is contained in a segment spanning -170/-107, resulted in a further 30% loss of the activity. Further removal of the proximal BBE (BBE1), which spans -107/-39, abolished greater than 95% of the basal expression. In addition, an A23187-inducible element (AIE) appeared to be associated with the BBE1. At least a six-fold inducibility remained as long as the BiP promoter retained the sequences -107/-39. Using an in vitro gel mobility shift assay, an A23187-inducible nuclear factor (AINF) was detected from NIH3T3 cells. DNA binding competition experiments indicate that the -107/-39 segment contains a sequence motif which interacts with this cellular factor. Further analysis showed that the two direct repeats, ranging -108/-73 and -72/-40, are the target for AINF binding. A 3-4 fold increase of the AINF binding to both repeated sequences was detected from induced cells. Similar results were also demonstrated in HeLa cells, suggesting that transcriptional control of BiP gene expression in mammalian cells is conserved. These findings also imply that the identified nuclear factor may be important in mediating transcriptional activation of the BiP gene.
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PMID:A direct-repeat sequence of the human BiP gene is required for A23187-mediated inducibility and an inducible nuclear factor binding. 148 Apr 70

The 5'-flanking region of protein kinase C (PKC) gamma gene was identified from a rat liver genomic library in a bacteriophage lambda Charon 4A. A 3.6-kilobase (kb) genomic fragment containing the 5'-flanking region, first exon, and first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 mapping and primer extension, was located 243 base pairs upstream from the translational initiation site. Promoter activity of a DNA segment spanning the 5'-flanking region was demonstrated by both in vitro transcription using HeLa cell nuclear extracts and chloramphenicol acetyltransferase assay by transfection of 293 cells with a PKC gamma-CAT fusion construct. Chloramphenicol acetyltransferase assay revealed that a fragment of about 0.16 kb from the transcriptional initiation site was sufficient for promoter activity in these cells, and the construct containing up to 1.6 kb from the cap site was expressed at a similar level. This promoter-active fragment contains several regions similar to defined transcriptional elements in other mammalian promoters, such as those for stimulatory protein 1 (Sp1), activator proteins 1 and 2 (AP1, AP2), c-myc, cAMP regulatory element-binding protein (CREB), and enhancer core (EnhC). Investigation of the genomic structure of PKC gamma gene may lead to the identification of cis-elements controlling tissue-specific and developmental stage-specific expression of PKC gamma.
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PMID:Characterization of the 5'-flanking region of the rat protein kinase C gamma gene. 224 72

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose synthesis and secretion by mesenchymal cells is regulated at the level of gene transcription by platelet-derived growth factor. To examine the transcriptional regulation of the TSP gene at the molecular level, a genomic clone containing the human TSP promoter and flanking sequence was isolated and characterized. A 3.8-kilobase pair (kb) DNA fragment containing the first three exons, the first two introns, and 2.2 kb of 5'-flanking region was sequenced, and the site of transcription initiation was determined by both primer extension and S1 nuclease mapping. Consensus sequences for several potential regulatory elements were found in the 5'-flanking sequence, including a TATA box consensus sequence, TTTAAAA, located 24 base pairs upstream from the transcription start site. A chimeric gene was constructed containing the first intron, the first exon, and 2.0 kb of 5'-flanking sequence of the TSP gene fused to the promoterless gene for chloramphenicol acetyltransferase. When transfected into COS-1 or NIH3T3 cells this gene construct was transcribed, indicating the presence of a functional promoter in the TSP sequence. Transient transfection studies using deletion mutants of this TSP-chloramphenicol acetyltransferase construct were performed to locate cis-acting regulatory sequences. The deletion of flanking sequence 5' to position -234 had little or no effect on transcriptional activity, whereas deletion of 5'-flanking sequence extending further in the 3' direction resulted in the gradual loss of transcriptional activity. The removal of the first intron resulted in a 4-fold decrease in transcript levels, indicating the presence of a cis-acting positive element(s) in the first intron of the human TSP gene. This element(s) was further localized to the region between position +576 and position +727.
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PMID:Characterization of the promoter region of the human thrombospondin gene. DNA sequences within the first intron increase transcription. 254 87

To gain insight into how transcription of the human p53 oncogene is controlled, we characterized the regulatory regions of the gene. A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5' end of the human p53 gene, as well as subfragments generated by restriction digests, was cloned upstream of the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and CAT activity was assayed in extracts of transfected cells. Two types of CAT vectors were used: Epstein-Barr virus oriP-derived constructs that were stably introduced into the human cell lines K562, Raji, and HL-60, and pSV0-CAT-derived constructs that were transiently introduced into the monkey cell line COS. By this approach we have identified two promoters for the human p53 gene. One promoter, p53P1, is located 100-250 bp upstream of the 218-bp noncoding first exon; a second, stronger promoter, p53P2, maps within the first intron. CAT activity and expression of CAT RNA indicate that p53P2 functions up to 50-fold more efficiently than p53P1. We conclude that the expression of the human p53 gene may be controlled by two promoters and that differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells.
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PMID:Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1. 283 31

Nucleotide sequences necessary to direct transcription of the gene encoding atrial natriuretic factor (ANF) in neonatal and fetal hearts have been defined by using expression of the prokaryotic marker gene chloramphenicol acetyltransferase (CAT) as a functional assay. Hybrid ANF-CAT genes were introduced into primary cultured cardiocytes by electroporation. A 3.4-kilobase (kb) fragment containing sequences on the 5' side of the ANF gene promoted significant CAT activity in atrial but not ventricular cardiocytes derived from 1-day-old rats. Deletion analysis of putative regulatory regions demonstrated that 2.4 kb of 5' ANF sequences were sufficient for high-level atrial transcription, whereas hybrid genes containing less than 700 base pairs of ANF sequences promoted less CAT activity. Cardiocytes derived from embryonic ventricles expressed the 3.4-kb ANF-CAT hybrid gene at levels comparable to atrial cells, suggesting that the nucleotide sequences controlling developmental regulation of ANF expression are contained in this 5' region. Nucleotide sequence analysis of this 3.6-kb region identified segments that may contribute to the regulated expression of the ANF gene.
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PMID:Cis-acting sequences that modulate atrial natriuretic factor gene expression. 296 98

The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451-462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CAAT box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.
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PMID:Characterization of the promoter region of the human MDR3 P-glycoprotein gene. 789 60

The 5'-flanking region of the human neurotrophin-3 (NT-3) gene was isolated from a human placental genomic library using the oligonucleotide corresponding to the 5'-noncoding region of the NT-3 cDNA as a probe. A 3.8 kbp genomic fragment containing the 5'-flanking region, the first exon and a portion of the first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 nuclease mapping, was located 27 bp downstream from the TATA-like sequence. Several plasmids, in which the NT-3 promoter regions were fused to the chloramphenicol acetyltransferase (CAT) gene, were constructed. Transient expression in human glioma Hs683 cells demonstrated that a fragment of about 0.1 kbp from the transcriptional initiation site was sufficient for promoter activity. While, in human plasma cell leukemia ARH77 cells, in which NT-3 mRNA was not detected, the region upstream from -65 functioned to silence CAT activity. It is suggested that this region contains the transcriptional regulatory element for the specific expression of the NT-3 gene.
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PMID:Identification of the functional regulatory region of the neurotrophin-3 gene promoter. 838 96

The mechanisms controlling the proliferation of astrocytes are of great interest but are not well defined. We have previously shown that the endogenous neuropeptides, endothelin-3 (ET-3), and atrial natriuretic peptide (ANP), modulate the proliferation of astrocytes through positively and negatively regulating the transcription of the immediate-early gene egr-1 which transactivates basic fibroblast growth factor (bFGF) by unknown mechanisms. In these studies, we determined the involvement of MAP kinase (Erk) activation by ET-3 in the transcription of egr-1, and the molecular determinants by which Egr-1 transactivates bFGF. Transfection of astrocytes with a mitogen-activated protein (MAP) kinase (MAPK) expression vector increased the transcription of a cotransfected egr-chloramphenicol acetyltransferase (CAT) construct 3-fold. This induction was totally abolished by a dominant negative MAPK mutant. A 3-fold induction of egr-CAT expression by ET-3 was significantly reduced by treatment with ANP, or a cotransfected dominant negative MAPK plasmid. Using mobility shift assays, we showed that ET-3 induced the expression of Egr-1 protein which bound specifically to several early growth-related protein (Egr-1) binding sites on the bFGF promoter, and that this effect was significantly reversed by treatment with ANP. We also found that the Sp1 transcriptional factor was bound at these same sites, but was not stimulated by ET-3. Deletion experiments indicated that only the site at -160 bp of the bFGF promoter was significant for bFGF transactivation by Egr-1. We conclude that the astrocyte mitogen, ET-3, stimulates egr-1 transcription through a MAP kinase (Erk) related mechanism, and that Egr-1 transactivates bFGF through a specific noncanonical, Egr-1 site on the promoter. ANP inhibits each of these steps, providing a pathway for its anti-proliferative action.
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PMID:Egr-1 activates basic fibroblast growth factor transcription. Mechanistic implications for astrocyte proliferation. 870 7

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.
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PMID:Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. 878 Nov 19


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