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)

We have compared the activities of mouse alpha-fetoprotein (AFP) enhancers I, II, and III with their minimal enhancer fragments (Mers) I, II, and III and with the entire 7-kilobase pair enhancer domain by transient expression assay in primary fetal mouse liver cells. The level of expression directed by the AFP promoter [p(-1009)AFPcat] alone is stimulated at least 10-fold by the entire AFP enhancer domain (-1009 to -6983). Enhancer I can drive the level of chloramphenicol acetyltransferase activity equivalent to that of the entire enhancer domain, whereas the increase in activity by enhancers II and III is significantly lower (1.5-fold). MersI, II, and III all mediate a greater increase in activity than their corresponding enhancer regions. The increase with MerI is 16-fold. Using DNase I protection analyses we identified 3 protein-binding regions in MerI; site Ia binds liver and brain nuclear proteins; site Ib binds liver, kidney, and brain nuclear proteins as well as purified C/EBP; site Ic binds liver and kidney nuclear proteins. Site-specific mutation of Ia, Ib, or Ic showed a 10-25% reduction in chloramphenicol acetyltransferase expression; deletion of the C/EBP-binding site in Ib showed a 45% reduction in activity and mutation of all 3 sites (Ia, Ib, and Ic) resulted in a 75% reduction in activity. Our studies indicate no single trans-acting factor is absolutely essential for enhancer activity, and that the enhancer activity of MerI is mediated via a combinatorial and additive mechanism.
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PMID:Functional analysis of the mouse alpha-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures. 137 27

Enhancer-like sequences have previously been identified in the promoter region of the mouse major histocompatibility complex (MHC) class I genes. We have screened for such sequences in and around a human MHC class I gene, HLA-B7. Various restriction fragments of the B7 gene were assayed for their ability to enhance transcription of a bacterial chloramphenicol acetyltransferase gene from a simian virus 40 promoter in transiently transfected mouse LTA cells. Our results demonstrate that enhancer activity is located in introns 3 and 5 as well as 5' to the transcription initiation site. RNase protection experiments corroborate the results. Preliminary experiments indicate that B7 enhancers are active in various cell types. The role of these enhancers in B7 gene expression is not known at present. We speculate that the position of the enhancer elements may be related to the occurrence of Hpa II tiny fragment islands.
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PMID:Multiple enhancer-like sequences in the HLA-B7 gene. 250 82

We have identified three elements in the noncoding region of human papillomavirus type 6 (HPV-6) that regulate transcription when assayed in recombinant plasmids containing the bacterial gene for chloramphenicol acetyltransferase. One was a silencer that reduced expression in both a species- and tissue-dependent manner. The second was an enhancer element that was tissue specific. The third was a weak promoter that showed some tissue specificity. These elements have been localized within the noncoding region by analysis of 5'-to-3' and 3'-to-5' deletions with two HPV-6 subtypes, HPV-6e and HPV-6g. HPV-6g differs from HPV-6e by the presence of an additional copy in tandem of a 136-base-pair (bp) sequence and by an 8-bp sequence containing a 3-bp deletion. Silencer activity, assayed in plasmids with the simian virus 40 minimum promoter which were transfected into NIH 3T3 cells, could not be overcome by the enhancer activity of the simian virus 40 72-bp repeats. The 413-bp fragment of A of HPV-6g showed silencer activity, while the corresponding HPV-6e fragment containing the 8-bp change did not. Enhancer activity of HPV-6g was localized to fragment C of 326 bp which contains the 136-bp repeat. Dot blot hybridizations reflected relative chloramphenicol acetyltransferase activities and demonstrated enhancer and silencer activities at the RNA level. Analysis of the interaction of these activities in naturally occurring variants should provide information on tissue specificity and regulation of gene expression of HPVs and may provide information on the mechanism of action of transcriptional regulatory elements in eucaryotic cells.
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PMID:Transcriptional regulatory elements in the noncoding region of human papillomavirus type 6. 284 82

We have cloned multiple copies of 72-base-pair (bp) repeat transcriptional enhancer element from simian virus 40 in plasmid vectors upstream from the bacterial chloramphenicol acetyltransferase gene under the control of the simian virus 40 early promoter. Two copies of the 72-bp repeat provided efficient activation of gene expression. Increasing the number of linked 72-bp units to four substantially improved the activation of gene expression, but further addition of enhancers diminished the activation of gene expression proportionally to the number of enhancers added. Enhancer regions containing 10 or more copies of the 72-bp sequence were very inefficient in gene activation. Plasmids containing such expanded enhancer regions also competed less efficiently in vivo for trans-acting enhancer-binding factors. These effects are specific for the enhancer element and are not produced by reiterations of the 21-bp promoter element. Multiple enhancer units placed upstream do not interfere with the accuracy of mRNA initiation directed by the simian virus 40 early promoter in these plasmid constructs but do severely inhibit the initiation of replication at the neighboring simian virus 40 origin of replication that overlaps the early promoter region. These results are consistent with the hypothesis that the structural alterations induced in the DNA by a large number of copies of the enhancer are not favorable for the activation of a linked gene or for the binding of factors believed to mediate the enhancement effect.
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PMID:Activation of gene expression is adversely affected at high multiplicities of linked simian virus 40 enhancer. 301 Feb 81

We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta-globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.
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PMID:Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences. 301 46

Using a short-term cotransfection system with recombinant chloramphenicol acetyltransferase (CAT) target genes and intact genes for regulatory proteins, we previously demonstrated that expression from the promoter-regulatory region of the gene for the immediate-early 175,000-molecular-weight (IE175K) protein of herpes simplex virus type 1 was subject to trans-acting effects by three different virus-encoded components. In the present work we have attempted to delineate the upstream cis-acting requirements within the IE175K promoter-regulatory region for stimulation by the late structural protein Vmw65, stimulation by the IE110K protein, and repression by its own gene product, the IE175K protein. Our results augment previous reports of others by demonstrating that a construct containing only the single TAATGARAT consensus sequence, TAATGGAAT, between -115 and -106 was efficiently induced by Vmw65. Deletion to -108 effectively abolished the response to Vmw65. However, this latter construct remained responsive to IE110K stimulation and was induced as efficiently as the parental construct which contained sequences to -1900. Furthermore, not only basal levels of expression, but also Vmw65 activation of the parental construct and deletion mutants delta 380, delta 330, delta 300, and delta 160 and IE110K-activated expression of the delta 108 construct were all subject to dominant repression by the IE175K protein. Finally, we show that expression from each of the deletions was open to stimulation by linkage to the simian virus 40 enhancer region. Enhancer-stimulated expression from each construct, including the -108 deletion, was efficiently repressed by the IE175K protein. In contrast, expression from the simian virus 40 enhancer when linked to its own promoter was unaffected by IE175K. These results place sequence requirements for both IE110K stimulation and IE175K autoregulation within the minimal promoter region -108 to +30, separate from the major requirements for Vmw65 activation located further upstream.
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PMID:Comparison of upstream sequence requirements for positive and negative regulation of a herpes simplex virus immediate-early gene by three virus-encoded trans-acting factors. 302 97

The upstream regulatory regions of human papillomavirus (HPV) types 1, 6b, 7, 11, 16, and 18, bovine papillomavirus type 1, and cottontail rabbit papillomavirus were cloned into transcriptional enhancer assay plasmids which carry the simian virus 40 early promoter lacking its own enhancer and the bacterial gene encoding chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT). Enhancer activity, reflected by CAT gene expression, was detected in all of the upstream regulatory regions tested only when the recombinants were cotransfected with plasmids which express an intact E2 open reading frame of HPV types 1 and 11 or bovine papillomavirus type 1. Each E2 protein stimulated the enhancer from the same virus and, to somewhat lesser degrees, also those from the heterologous viruses. Hence, the enhancer and the E2 protein are functionally conserved among papillomaviruses. There was some nonreciprocity in the extent of trans-activation in heterologous E2-enhancer interactions. Primer extension analyses demonstrated that the E2 proteins increased the abundance of CAT gene mRNA. Tandem multiplication of the HPV type 11 enhancer sequence dramatically increased its response to E2 stimulation; this is possibly relevant to the pathogenicity of papillomaviruses.
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PMID:Enhancers and trans-acting E2 transcriptional factors of papillomaviruses. 303 19

Enhancer elements regulating the neuronal gene, tyrosine hydroxylase (TH), were identified in TH-expressing peripheral nervous system PATH and central nervous system CATH cell lines. Mutational analysis in which rat TH 5'-flanking sequences directed chloramphenicol acetyltransferase (CAT) reporter gene expression demonstrated that mutating the cyclic AMP response element (CRE) at -45 base pair reduced expression by 80-90%. A CRE linked to an enhancerless TH promoter fully supported expression. Cotransfection of a dominant-negative CREB protein reduced expression 50-60%, suggesting that the CRE is bound by CREB or a CREB dimerization partner. Although mutating the AP1/dyad (AD) element at -205 base pair only modestly reduced CAT levels, AD minimal enhancer constructs gave 45-80% of wild type expression when positioned at -91 or -95. However, in its native context at -205, the AD could not support expression. In contrast, a CRE, moved from its normal position at -45 to -206, gave full activity. These results indicate that the CRE is critical for TH transcription in central nervous system CATH and peripheral nervous system PATH cells, whereas the AD is less important and its enhancer activity is context-and/or position-dependent. These results represent the first attempts to map regulatory elements directing TH expression in central nervous system cell lines.
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PMID:The cyclic AMP response element directs tyrosine hydroxylase expression in catecholaminergic central and peripheral nervous system cell lines from transgenic mice. 766 71

Expression of the human alpha 2A-adrenergic receptor gene is induced by cAMP. The present studies were designed to define potential cAMP-responsive enhancer elements (CREs) in the promoter region of this gene. Regions from the 5'-flanking sequences of the gene were placed in a promoterless vector with a chloramphenicol acetyltransferase (CAT) reporter gene, and cAMP-stimulated CAT activity was assayed in transfected JEG-3 placental carcinoma cells. Enhancer activity responsive to cAMP was located in DNA sequences both upstream and downstream from the endogenous promoter region. Within the upstream sequences there is a putative "core sequence" homologous to the eight base CRE consensus palindrome, but this region did not function independently as a CRE enhancer; additional upstream sequences were required to provide significant enhancer activity in response to cAMP. Regulation of expression of the alpha 2A-adrenergic gene by cAMP is complex and involves multiple and likely novel DNA sequences.
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PMID:Enhanced transcription of the human alpha 2A-adrenergic receptor gene by cAMP: evidence for multiple cAMP responsive sequences in the promoter region of this gene. 801 30

The androgen receptor (AR) is a member of the steroid receptor superfamily that binds to the androgen response element to regulate target gene transcription. AR may need to interact with some selected coregulators for maximal or proper androgen function. Here we report the isolation of a new AR coregulator with a calculated molecular mass of 267 kDa named the androgen receptor-associated protein 267-alpha (ARA267-alpha). ARA267-alpha contains 2427 amino acids, including one Su(var)3-9, Enhancer-of-zeste, and Trithorax (SET) domain, two LXXLL motifs, three nuclear translocation signal (NLS) sequences, and four plant homeodomain (PHD) finger domains. Northern blot analyses reveal that ARA267-alpha is expressed predominantly in the lymph node as 13- and 10-kilobase transcripts. HepG2 is the only cell line tested that does not express ARA267-alpha. Yeast two-hybrid and glutathione S-transferase pull-down assays show that both the N and C terminus of ARA267-alpha interact with the AR DNA- and ligand-binding domains. Unlike other coregulators, such as CBP, which enhance the interaction between the N and C terminus of AR, we found that ARA267-alpha had little influence on the interaction between the N and C terminus of AR. Luciferase and chloramphenicol acetyltransferase assays show that ARA267-alpha can enhance AR transactivation in a dihydrotestosterone-dependent manner in PC-3 and H1299 cells. ARA267-alpha can also enhance AR transactivation with other coregulators, such as ARA24 or PCAF, a histone acetylase, in an additive manner. Together, our data demonstrate that ARA267-alpha is a new AR coregulator containing the SET domain with an exceptionally large molecular mass that can enhance AR transactivation in prostate cancer cells.
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PMID:Identification and characterization of a novel androgen receptor coregulator ARA267-alpha in prostate cancer cells. 1150 67


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