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'-terminal region of the rat gene for the neuron-specific phosphoprotein, synapsin I, was isolated and sequenced. It comprises 1472 nucleotides (nt) of 5'-flanking sequence, 507 nt of the first exon, and 242 nt of the first intron. A single transcription start site was mapped by primer extension and S1 nuclease analysis. A sequence of 340 nt upstream from the transcription start site and the first exon are G+C-rich and enriched in CpG dinucleotides, resembling a CpG island. The 5'-flanking sequence lacks TATA and CAAT consensus elements but contains a consensus motif for the cAMP-responsive element. Furthermore, we notice two potential consensus motifs which are also found in corresponding positions in the genes for the nerve growth factor receptor and the 68-kDa neurofilament protein. The 5'-terminal region of the human synapsin I gene was also cloned and sequenced. A high degree of sequence conservation between rat and human is found in the upstream 340 nt that coincides precisely with the G+C-rich domain and includes the consensus elements, and throughout the first exon including the untranslated sequence. Sequence conservation is also observed further upstream and at the beginning of the first intron. In a transient chloramphenicol acetyltransferase expression assay, 5'-flanking sequences of the rat synapsin I gene function as strong promoters in neuroblastoma cells, but not in fibroblastoid cells. 225 nt of 5'-flanking sequence and 105 nt of 5'-untranslated sequence are sufficient for cell-type specific transcription in this assay.
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PMID:The 5'-flanking region of the synapsin I gene. A G+C-rich, TATA- and CAAT-less, phylogenetically conserved sequence with cell type-specific promoter function. 211 19

We report the identification and characterization of the cis-acting elements responsible for the expression of the rat cholecystokinin (CCK) gene. Deletion mutations were constructed by linking variable amounts of the 5'-flanking region of the CCK gene to the bacterial chloramphenicol acetyltransferase reporter gene. The transcriptional activity of the CCK promoter deletion constructs was measured by monitoring chloramphenicol acetyltransferase enzyme activity after transient transfections. It is shown that sequences within 102 base pairs of the cap site are required for the expression from this promoter. This region contains a sequence that is identical to the -296 element of the human c-fos gene and is homologous with the polyoma enhancer and the cAMP- and 12-O-tetradecanoylphorbol-13-acetate-responsive elements described for several genes. In addition, the -119 to -81 fragment of the CCK promoter contains a transcriptional enhancer that potentiates the transcription from the herpes simplex virus thymidine kinase promoter in a position- and orientation-independent manner. DNase I protection and gel retardation experiments indicated the ability of several trans-acting factors found in nuclear extracts to bind specifically to regions of the CCK promoter. In particular, two complexes formed adjacent to the CCK enhancer region. One complex, CCK-1a, formed with sequences 5' to the enhancer whereas the other complex, CCK-1b, formed with the sequences identified by DNase I footprinting, 3' to the enhancer. Oligonucleotide competition experiments indicated that these complexes are formed by the same transacting factor or factors with similar binding specificities.
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PMID:A transcriptional enhancer essential for the expression of the rat cholecystokinin gene contains a sequence identical to the -296 element of the human c-fos gene. 211 25

The promoter region of the poly(ADP-ribose) polymerase gene has been isolated using a Sau3AI genomic library derived from human leukocyte. It lacks typical transcriptional regulatory elements such as TATA and CAAT boxes, but it contains two potential Sp1 binding sites and three putative AP-2 binding elements. The region up to nucleotide position-99 in relation to the predominant transcriptional initiation site exhibits promoter activity as judged by chloramphenicol acetyltransferase assay and the activity is enhanced both by cAMP and by phorbol ester. Northern blot and Western blot analyses have revealed that expression of the polymerase gene is also stimulated by both of these compounds in cultured HeLa cells. Southern blot hybridization of genomic DNA separately digested with various endonucleases gives a discrete single band in each case when the 5'-untranslated region of the polymerase cDNA is used as a probe. These results indicate that poly(ADP-ribose) polymerase is encoded by a unique gene whose expression is regulable by cAMP and by phorbol ester.
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PMID:Human poly(ADP-ribose) polymerase gene. Cloning of the promoter region. 212 69

Fibronectin (FN) mRNA levels increased when quiescent cells (serum starved) were stimulated to undergo the G0/G1 transition by the addition of 20% given fetal calf serum to the media. The 5'-flanking region of the FN gene (position +69 to -510 base pairs (bp] was fused to the coding region of the chloramphenicol acetyltransferase (CAT), and the fusion gene was used in transfection assays. Expression of FNCAT increased on serum treatment indicating that the region of the FN gene between positions +69 and -510 bp mediated serum responsiveness. Deletion of FN gene 5'-flanking sequences from position -510 to -122 bp eliminated serum responsiveness suggesting that an element between these positions was mediating the effect. Sequences between positions -122 and -510 bp of the FN gene were able to confer serum responsiveness on a herpes virus thymidine kinase promoter-CAT fusion gene (TKCAT) when the FN gene sequences were cloned upstream of TKCAT. The ability to confer serum responsiveness on TKCAT was retained with a smaller 100-bp sequence (position -122 to -222 bp). Both a cAMP response element (position -170 bp) and a nuclear factor-1 binding site (position -155 bp) have been identified within this sequence (Dean, D. C., Blakeley, M. S., Newby, R. F., Ghazal, P., Hennighausen, L., and Bourgeois, S. (1989) Mol. Cell. Biol. 9, 1498-1506). The cAMP response element was serum-responsive when cloned upstream of TKCAT or a minimal FN promoter (deleted to position -56 bp) while the nuclear factor-1 binding site was unresponsive. Therefore, the cAMP regulatory element (CRE) is the serum-responsive element between position -122 and -222 bp. Serum-induced binding of proteins to the CRE was detected in gel retardation assays with extracts from cell lines where FN expression was serum-responsive. However, no serum-induced binding was detected with extracts from the JEG-3 cell line where FN expression was not serum-responsive. Serum-induced binding occurred rapidly, within 15 min, and did not require protein synthesis. The decay of serum-induced binding was relatively slow as increased binding was still detectable 24 h after removal of serum. The CRE also mediates transcriptional stimulation by cAMP, but unlike serum stimulation increased CRE binding activity was not detectable in extracts from cAMP-treated cells (Dean, D. C., Blakeley, M. S., Newby, R. F., Ghazal, P., Hennighausen, L., and Bourgeois, S. (1989) Mol. Cell. Biol. 9, 1498-1506).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serum stimulation of fibronectin gene expression appears to result from rapid serum-induced binding of nuclear proteins to a cAMP response element. 213 58

Transforming growth factor beta 3 (TGF-beta 3) has been cloned from humans, chickens, pigs, and mice. Although the specific in vivo roles of this form of TGF-beta are unknown, the pattern of embryonic and tissue-specific expression of TGF-beta 3 suggests that it is involved in embryogenesis and cell differentiation. We have cloned and sequenced the TGF-beta 3 5'-flanking region to study the transcriptional regulation of this gene. Characterization of the 5'-flanking region showed a 1104-base pair 5'-untranslated region, a TATA box 21 bp upstream from the transcription start site, and cAMP-responsive element (CRE) and AP-2 binding site consensus sequences starting at 12 and 24 bp, respectively, upstream from the TATA box. Promoter fragments were cloned into a chloramphenicol acetyltransferase reporter plasmid to study functional activity. Basal transcriptional activity of the promoter was regulated by multiple upstream elements including the CRE and the AP-2 site. The CRE was important for both basal and forskolin induction of promoter activity. The TGF-beta 3 promoter was found to be strikingly dissimilar to the TGF-beta 1 promoter. Since the TGF-beta s have activity in promoting or inhibiting proliferation and differentiation of multiple cell types, it seems likely that the differential and tissue-specific transcriptional regulation of these genes is of fundamental importance in the induction and maintenance of differentiated cell types in various tissues.
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PMID:Structural and functional characterization of the transforming growth factor beta 3 promoter. A cAMP-responsive element regulates basal and induced transcription. 214 70

Analogues of cAMP have been reported to increase insulin mRNA levels in normal rat beta-cells and hamster insulinoma cells (HIT). To define the mechanisms by which cAMP modulates insulin gene expression, we first investigated its effects on the transcriptional rate of the insulin gene in HIT cells. Nuclear run-on assays revealed a 4-fold increase in transcription observed as early as 1 h after stimulation. To characterize the cis-acting sequences of the rat insulin I gene promoter and the trans-acting factors mediating the cAMP effect on insulin gene transcription, we constructed DNA plasmids containing various lengths of the rat insulin I gene 5'-flanking region linked to the bacterial reporter gene, chloramphenicol acetyltransferase (CAT). Studies of the transcriptional activity of 5'-deletionally and pointly mutated plasmids after transfection into HIT cells revealed the presence of a cAMP-responsive element (CRE), TGACGTCC, between -177 and -184 relative to the transcriptional start site, whose sequence closely matches the previously defined CREs, present in cAMP-responsive genes. Gel retardation and Southwestern assays identify a protein of molecular weight approximately 43,000, binding specifically to the insulin CRE. We conclude that the rat insulin I gene is regulated by cAMP through a CRE and that the nuclear protein interacting with it might be similar or identical to the previously purified cAMP-responsive protein, CREB.
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PMID:Functional characterization of a cAMP-responsive element of the rat insulin I gene. 215 35

cAMP regulates the expression of several genes by activation of a promoter consensus sequence which functions as a cAMP-response element. Evidence indicated that this is accomplished via cAMP dissociation of cAMP-dependent protein kinase into its regulatory (R) and catalytic (C) subunits. Our investigations of the role of these two subunits in gene expression provide direct and quantitative evidence that the C subunit is required for cAMP stimulation of the cAMP-response element in the vasoactive-intestinal-peptide gene in rat pheochromocytoma cells. After cotransfection of a metallothionein-regulated C-subunit expression vector (pCEV) and a vasoactive-intestinal-peptide--chloramphenicol acetyltransferase construct containing a cAMP-response element, we could demonstrate expression of transfected C-alpha-subunit mRNA (truncated size 1.7 kb) by Northern blot and a concentration-dependent C subunit stimulation of chloramphenicol acetyltransferase activity. Basal activity was stimulated 12- and 50-fold by pCEV (30 micrograms), in the absence and presence, respectively, of Zn2+. Metallothionein-regulated expression of C was demonstrated by results that showed a 2-4-fold increase in chloramphenicol acetyltransferase activity in the presence versus the absence of 90 microM Zn2+. In contrast, overexpression of the R-II beta regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R-II beta transfected together with C (ratio 2:1 and 4:1) inhibited the stimulation by the C subunit 70% and 90% respectively. Our results indicate that transfection of cAMP-dependent protein kinase subunits results in functional expression of both C-alpha and R-II beta subunits. Expression of the C subunit mediated cAMP-regulated gene expression but this expression could be inhibited by cotransfected R-II beta subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP-dependent protein kinase.
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PMID:Regulation of gene expression by transfected subunits of cAMP-dependent protein kinase. 215 96

To delineate cis-acting regulatory elements of the human elastin gene, several elastin promoter region/chloramphenicol acetyltransferase reporter gene constructs were developed. The spectrum of inserts, spanning from -2260 to +2, was shown to contain several SP-1 and AP2 binding sites, as well as putative glucocorticoid, cAMP, and 12-O-tetradecanoylphorbol-13-acetate responsive elements. Assay of promoter activity in transient transfections of rat aortic smooth muscle cells, human skin fibroblasts, HT-1080 human fibrosarcoma cells, HeLa cells, or mouse NIH-3T3 cells allowed delineation of several functional subregions within 2.26 kilobases of the 5'-flanking DNA. The results suggest that the basic promoter element resides within the region -128 to -1, and the 5'-flanking DNA contains several functional regulatory subregions. Also, the regulatory function of three putative SP-1 binding sites was demonstrated by transfections with a plasmid devoid of such sequences. These findings attest to the complexity of transcriptional regulation of the elastin gene.
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PMID:Deletion analyses of 5'-flanking region of the human elastin gene. Delineation of functional promoter and regulatory cis-elements. 216 Sep 83

By utilizing chimeric genes constructed from 5'-flanking sequences of the human CYP21B (P-450C21) gene and reporter genes (chloramphenicol acetyltransferase or rabbit beta-globin), a 34-nucleotide sequence has been found to be required for cAMP-dependent transcription. This sequence (-129/-96 base pairs) shows no homology to that of the consensus (CRE) cAMP-regulatory element. Gel retardation analysis shows that a protein-DNA complex is formed between this DNA sequence and nuclear proteins from mouse adrenal Y1 tumor cells or bovine adrenal cortical cells or human fetal adrenal tissue and that formation of this complex cannot be competed by DNA containing the consensus CRE sequence. Even though cAMP-enhanced accumulation of P-450C21 mRNA in primary cultures of bovine adrenocortical cells is inhibited by the protein synthesis inhibitor, cycloheximide, reporter gene transcription enhanced by the cAMP-responsive -129/-96-base pair fragment of the human CYP21B gene is not. We conclude that cAMP-dependent transcription of the human P-450C21 gene (CYP21B), an event required for maintenance of optimal steroidogenic capacity in the adrenal cortex, involves a stable transcription factor(s) distinct from the CRE-binding protein. Furthermore the cAMP-dependent cis-regulatory element of the human P-450C21 gene is distinct from those found associated with the other steroid hydroxylase genes, 17 alpha-hydroxylase cytochrome P-450, cholesterol side chain cleavage cytochrome P-450, and 11 beta-hydroxylase cytochrome P-450, suggesting that each of these genes may require its own set of specific transcription factors for cAMP-dependent regulation.
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PMID:cAMP-dependent transcription of the human CYP21B (P-450C21) gene requires a cis-regulatory element distinct from the consensus cAMP-regulatory element. 216 43

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.
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PMID:Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells. 216 40


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