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 gene coding for the gamma chain of human fibrinogen was characterized for its promoter activity. Reporter gene studies using chloramphenicol acetyltransferase as the indicator along with mutations in the DNA showed that a TATA-like sequence (-20 to -23 base pairs (bp)), a CAAT-like sequence (-54 to -57 bp), and an upstream stimulatory factor (USF) binding site (-66 to -77 bp) constitute a minimal promoter that mediates liver-specific expression of the gene. Electrophoretic gel mobility assays and antibody binding studies confirmed the interaction of USF with the binding site. An IL-6 responsive element with a sequence of CTGGAA located at -301 to -306 bp was shown to be a functional element in the IL-6 response. In contrast to the IL-6 responsive elements in the human alpha- and beta-fibrinogen genes, the element in the gene for the gamma chain of human fibrinogen was unaffected by the presence or elevated levels of the beta or delta isoforms of the CCAAT/enhancer binding proteins. A negative element with sequence homology to several silencer elements was also identified in the region of -348 to -390 bp of the gene for the gamma chain of human fibrinogen. A comparison of the regulatory elements in the genes coding for all three chains in fibrinogen is also presented.
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PMID:Characterization of the 5'-flanking region of the gene for the gamma chain of human fibrinogen. 749 36

Progression through the somatic cell cycle requires the temporal regulation of cyclin gene expression and cyclin protein turnover. One of the best-characterized examples of this regulation is seen for the B-type cyclins. These cyclins and their catalytic component, cdc2, have been shown to mediate both the entry into and maintenance of mitosis. The cyclin B1 gene has been shown to be expressed between the late S and G2 phases of the cell cycle, while the protein is degraded specifically at interphase via ubiquitination. To understand the molecular basis for transcriptional regulation of the cyclin B1 gene, we cloned the human cyclin B1 gene promoter region. Using a chloramphenicol acetyltransferase reporter system and both stable and transient assays, we have shown that the cyclin B1 gene promoter (extending to -3800 bp relative to the cap site) can confer G2-enhanced promoter activity. Further analysis revealed that an upstream stimulatory factor (USF)-binding site and its cognate transcription factor(s) are critical for expression from the cyclin B1 promoter in cycling HeLa cells. Interestingly, USF DNA-binding activity appears to be regulated in a G2-specific fashion, supporting the idea that USF may play some role in cyclin B1 gene activation. These studies suggest an important link between USF and the cyclin B1 gene, which in part explains how maturation promoting factor complex formation is regulated.
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PMID:Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter. 773 59

The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes, as a linear array, two accessory factor binding sites (AF1 and AF2) and two glucocorticoid receptor binding sites. All of these elements are required for a complete glucocorticoid response. AF1 and AF2 also partially account for the response of the PEPCK gene to retinoic acid and insulin, respectively. A second retinoic acid response element was recently located just downstream of the GRU. In this study we show that mutation of the 3' half-site of this element results in a 60% reduction of the glucocorticoid response of PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion constructs in transient transfection assays, thus the half-site is now termed AF3. A variety of assays were used to show that chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds specifically to AF3 and that upstream stimulatory factor (USF) binds to an E-box motif located 2 base pairs downstream of AF3. Mutations of AF3 that diminish binding of COUP-TF reduce the glucocorticoid response, but mutation of the USF binding site has no effect. The functional roles of AF1, AF2, and AF3 in the glucocorticoid response were explored using constructs that contained combinations of mutations in all three elements. All three elements are required for a maximal glucocorticoid response, and mutation of any two abolish the response.
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PMID:The orphan receptor COUP-TF binds to a third glucocorticoid accessory factor element within the phosphoenolpyruvate carboxykinase gene promoter. 894 35

A 69-base pair (bp) (-581/-513) fragment derived from human transcobalamin II distal promoter constructed upstream of a chloramphenicol acetyltransferase reporter gene demonstrated high bidirectional promoter activity in transfected epithelial Caco-2 cells. DNase I footprinting, gel mobility shift, supershift, and mutagenesis studies with the 69-bp fragment demonstrated that a GC box (-568/-559) and an E box (-523/-528), which interacted with Sp1/Sp3 and USF1/USF2 (where USF is upstream stimulatory factor), respectively, were required for the full transcriptional activity of this fragment. Whereas mutations in the GC box reduced the promoter activity by 50%, mutations in the E box alone or in both the E box and GC box resulted in 90% loss of transcriptional activity. The essential role of the E box in the bidirectional promoter activity was further demonstrated by transient transfection in Caco-2, K-562, and HeLa cells using a 29-bp (-541/-513) fragment that contained only the E box. Based on these results we suggest that 1) the E box is essential for both the GC box-dependent and -independent promoter activity of the 69-bp fragment, 2) cooperative interactions between Sp1/Sp3 and USFs are required for the full activation of the 69-bp promoter activity, and 3) the single E box is able to mediate bidirectional transcription in transfected cells in the absence of an obvious TATA box or a known initiator element.
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PMID:A 69-base pair fragment derived from human transcobalamin II promoter is sufficient for high bidirectional activity in the absence of a TATA box and an initiator element in transfected cells. Role of an E box in transcriptional activity. 977 37

E-box/basic helix-loop-helix (bHLH)-dependent regulation of promoters for skeletal muscle-specific genes is well established, but similar regulation of smooth muscle-selective promoters has not been reported. Using transient transfection assays of smooth muscle alpha-actin (SMalphaA) promoter-chloramphenicol acetyltransferase (CAT) reporter constructs in rat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, we identified two activator elements, smE1 and smE2, with sequences corresponding to E-box (5'-CAnnTG-3') motifs. In L6 myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completely abolished promoter activity. In contrast, mutation of smE1 and smE2 was required to reduce promoter activity in SMCs. Supershift analyses identified a myogenin-containing complex as the predominant smE1 and smE2 binding activity in skeletal muscle, and myogenin overexpression transactivated the promoter. Supershift analyses with SMC extracts demonstrated that the bHLH protein upstream stimulatory factor (USF) bound smE1, and USF overexpression transactivated the promoter in an smE1-dependent manner. In summary, our results provide novel evidence implicating E-box elements in directing expression of the SMalphaA promoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.
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PMID:Differential activation of the SMalphaA promoter in smooth vs. skeletal muscle cells by bHLH factors. 1036 6

We previously reported that 2.1 kilobase pairs of the 5'-flanking sequence are sufficient for tissue-specific and hormonal/metabolic regulation of the fatty-acid synthase (FAS) gene in transgenic mice. We also demonstrated that the -65 E-box is required for insulin regulation of the FAS promoter using 3T3-L1 adipocytes in culture. To further define sequences required for FAS gene expression, we generated transgenic mice carrying from -644, -444, -278, and -131 to +67 base pairs of the rat FAS 5'-flanking sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Similar to the expression observed with -2100-FAS-CAT transgenic mice, transgenic mice harboring -644-FAS-CAT and -444-FAS-CAT expressed high levels of CAT mRNA only in lipogenic tissues (liver and adipose tissue) in a manner identical to the endogenous FAS mRNA. In contrast, -278-FAS-CAT and -131-FAS-CAT transgenic mice did not show appreciable CAT expression in any of the tissues examined. When previously fasted mice were refed a high carbohydrate, fat-free diet, CAT mRNA expression in transgenic mice harboring -644-FAS-CAT and -444-FAS-CAT was induced dramatically in liver and adipose tissue. The induction was virtually identical to that observed in -2100-FAS-CAT transgenic mice and to the endogenous FAS mRNA. In contrast, -278-FAS-CAT transgenic mice showed induction by feeding, but at a much lower magnitude in both liver and adipose tissue. The -131-FAS-CAT transgenic mice did not show any CAT expression either when fasted or refed a high carbohydrate diet. To study further the effect of insulin, we made these transgenic mice insulin-deficient by streptozotocin treatment. Insulin administration to the streptozotocin-diabetic mice increased CAT mRNA levels driven by the -644 FAS and -444 FAS promoters in liver and adipose tissue, paralleling the endogenous FAS mRNA levels. In the case of -278-FAS-CAT, the induction observed was at a much lower magnitude, and deletion to -131 base pairs did not show any increase in CAT expression by insulin. This study demonstrates that the sequence requirement for FAS gene regulation employing an in vitro culture system does not reflect the in vivo situation and that two 5'-flanking regions are required for proper nutritional and insulin regulation of the FAS gene. Cotransfection of the upstream stimulatory factor and various FAS promoter-luciferase constructs as well as in vitro binding studies suggest a function for the upstream stimulatory factor at both the -65 and -332 E-box sequences.
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PMID:Two 5'-regions are required for nutritional and insulin regulation of the fatty-acid synthase promoter in transgenic mice. 1074 93

The transcription of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis, is dramatically induced by fasting/refeeding and insulin. We reported that upstream stimulatory factor binding to the -65 E-box is required for induction of the FAS transcription by insulin in 3T3-L1 adipocytes. On the other hand, we recently found that two upstream 5' regions are required for induction in vivo by fasting/refeeding and insulin; one at -278 to -131 albeit at a low level, and the other at -444 to -278 with an E-box at -332 where upstream stimulatory factor functions for maximal induction. Here, we generated double transgenic mice carrying the chloramphenicol acetyltransferase reporter driven by the various 5' deletions of the FAS promoter region and a truncated active form of the sterol regulatory element (SRE) binding protein (SREBP)-1a. We found that SREBP participates in the nutritional regulation of the FAS promoter and that the region between -278 and -131 bp is required for SREBP function. We demonstrate that SREBP binds the -150 canonical SRE present between -278 and -131, and SREBP can function through the -150 SRE in cultured cells. These in vivo and in vitro results indicate that SREBP is involved in the nutritional induction of the FAS promoter via the -278/-131 region and that the -150 SRE is the target sequence.
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PMID:Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element. 1096 28

Somatotropin (ST) antagonizes insulin stimulation of fatty acid synthase (FAS) enzyme activity and gene transcription in adipocytes. Previous studies have shown that an insulin response element (IRE) is located in the proximal region of the FAS promoter (-71 to -50) and upstream stimulatory factor (USF) 1 binds to this IRE. The present study was conducted to initially evaluate whether there is a somatotropin response element (STRE) in the 5'flanking region of the FAS gene and to determine whether USF1 mediates the effect of ST on FAS gene transcription in 3T3-F442A adipocytes. Two 5' deletion FAS promoter constructs (pFAS-CATS4 and pFAS-CAT5), which contain the 5' flanking sequences of the rat FAS gene at -112 to +65 and -2195 to +65, respectively, were stably transfected into 3T3-F442A preadipocytes. Insulin stimulated chloramphenicol acetyltransferase (CAT) activity 1.7- and 4.7-fold (P < 0.05) in 3T3-F442A adipocytes transfected with pFAS-CATS4 and pFAS-CAT5, respectively. In contrast, bovine somatotropin (bST) attenuated the stimulatory effect of insulin on CAT activity by approximately 60% (P < 0.05) in both constructs. When 3T3-F442A adipocytes were treated with insulin (10 ng/mL) or insulin (10 ng/mL) plus bST (100 ng/mL) for 24, 48, or 72 h, neither insulin nor bST significantly affected USF1 mRNA levels. When human USF1 (hUSF1) cDNA probe was used, however, insulin increased the abundance of an unidentified transcript (named hUSF1-like mRNA) 11- to 25-fold (P < 0.05) and ST decreased the stimulatory effect of insulin on hUSF1-like mRNA levels by 50 to 90% (P < 0.05). Western blot analyses of nuclear extracts from cells treated with insulin (10 ng/mL) or insulin (10 ng/mL) plus bST (100 ng/mL) for 48 h demonstrated that the abundance of USF1 was not affected by insulin or ST. Furthermore, electrophoretic mobility shift analyses (EMSA) of nuclear extracts revealed that neither insulin nor ST had an effect on the binding of USF1 to the IRE. These results suggest that a STRE may be located within the first 112 bp of the FAS promoter and that USF1 does not directly mediate the effect of ST on transcription of the FAS gene in 3T3-F442A adipocytes.
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PMID:Transcriptional regulation of fatty acid synthase gene by somatotropin in 3T3-F442A adipocytes. 1158 20

Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived beta TC-3 cells revealed that multiple promoter regions, located between -306 and -97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans -acting factor-binding sites in the IGRP promoter that were identified in beta TC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans -acting factor-binding sites located between -97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the -306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 (neurogenic differentiation/beta-cell E box transactivator 2) and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.
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PMID:Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression. 1254 Feb 93

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