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 expressed in the yeast Saccharomyces cerevisiae a full-length poliovirus cDNA clone under the control of the GAL10 promoter to better characterize the effect of poliovirus on host cell metabolism. We find that yeast cells are unable to translate poliovirus RNA in vivo and that this inhibition is mediated through the 5' untranslated region of the viral RNA. The in vivo inhibition of translation of poliovirus RNA and P2CAT RNA (which contains the 5' untranslated region fused upstream of the bacterial chloramphenicol transferase gene) can be mimicked in vitro in yeast translation lysates. In fact, a trans-acting inhibitor present in yeast lysates can inhibit translation of either poliovirus or P2CAT RNA in HeLa cell translation lysates. In contrast, when the inhibitor is added to translations programmed with chloramphenicol acetyltransferase RNA, yeast prepro-alpha-factor RNA, or an RNA containing the internal ribosome entry site of encephalomyocarditis virus, no inhibition is seen. The inhibitory activity has been partially purified by DEAE-Sephacel chromatography. The partially purified inhibitor is heat stable, escapes phenol extraction, is resistant to proteinase K and DNase I treatment, and is sensitive to RNase A digestion, suggesting that the inhibitor is an RNA. In an in vitro translation assay, the inhibitory activity can be overcome by increasing the concentration of HeLa cell lysate but not P2CAT RNA, suggesting that the inhibitor interacts (directly or indirectly) with one or more components of the HeLa cell translational machinery rather than with the viral RNA.
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PMID:Yeast cells are incapable of translating RNAs containing the poliovirus 5' untranslated region: evidence for a translational inhibitor. 130 48

As an initial step toward understanding the transcriptional regulation of cholesterol 7 alpha-hydroxylase (CYP7) in man, we isolated and functionally characterized the 5'-flanking region of the human CYP7 gene. The nucleotide sequences of the first exon and 1.6 kb preceding the exon were determined and found to contain a TATA box at position -30, a modified CAAT box at position -92, three potential hepatocyte nuclear factor 3 (HNF-3) recognition sites at nucleotides -316, -288, and -255, respectively, and a modified sterol response element at position -271. DNA sequences containing 1.3 kb of the 5'-flanking region and 29 nucleotides of the first exon were linked to the chloramphenicol acetyltransferase gene and transiently transfected into several cell lines. Promoter activity was very strong in the human hepatoma cell line HepG2 but absent in cells of nonhepatic origin. Mutational analysis of the promoter identified several regions that function in the transcriptional regulation of CYP7. Introduction of a fragment containing the region from -432 to -220 upstream of a heterologous promoter, in either orientation, resulted in a tremendous stimulation of activity in HepG2 cells. DNase I footprint analysis identified three regions within this fragment which were protected from digestion. The overexpression of HNF-3 in HepG2 cells resulted in a 4-fold stimulation of CYP7 transcriptional activity. We suggest that the region between -432 and -220 functions as a cell-specific enhancer whose activity is controlled, in part, by HNF-3.
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PMID:Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene. 131 51

Factor X is a vitamin K-dependent glycoprotein that plays an essential role in both the intrinsic and extrinsic pathways of blood coagulation. Studies on a recombinant lambda phage containing the 5'-flanking region of the human factor X gene showed that the factor X gene was linked to and was located at the 3' end of the factor VII gene: the initiation codon of the factor X gene was 2823 base pairs (bp) downstream from the polyadenylation site of the factor VII gene. This 2.8-kilobase intergenic region, and progressively deleted fragments of it, was fused to the chloramphenicol acetyltransferase gene, and transient expressions in HepG2 cells, human fibroblasts, and Chinese hamster ovary cells were measured. A liver-specific promoter element, FXP1-binding site, essential for hepatocyte-specific transcription was identified. This promoter sequence, further localized to -63 to -42 bp in DNase I footprint studies, was homologous to LF-A1 or hepatic nuclear factor-4 recognition sequence and was equally functional in the normal and inverse orientations. FXP1 site bound to nuclear protein(s) from HepG2 cells and complex formation was partially abolished by the presence of duplex oligonucleotides containing liver factor-A1 or hepatic nuclear factor-4-binding sequences. Two additional positive elements located upstream of the promoter region, spanning from -215 to -149 bp (FXP2 site), and -457 to -351 bp (FXP3 site), were also established by reporter gene assays.
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PMID:Liver-specific expression of the gene coding for human factor X, a blood coagulation factor. 131 96

The purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP).
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PMID:Transactivation of the major capsid protein gene of herpes simplex virus type 1 requires a cellular transcription factor. 131 6

The ability of the human insulin receptor promoter to direct expression of a linked chloramphenicol acetyltransferase gene was assessed in transient transfections into HepG2 and Hela cells. A 5'-deletional analysis of the promoter showed that regions between -646 and -489 were important for the activity of the proximal promoter. In addition, a possible negative regulatory element was identified between -1311 and -877 and a positive element between -1823 and -1311. DNase I footprint and gel retardation analysis showed that multiple factors bind to the human insulin receptor promoter. In particular, DNase I protection patterns were observed over the Sp1 sites at -620 to -599 and -438 to -392, a TC box at -533, four homopyrimidine/homopurine sites clustered around -1150, and a site at -1420 that contains the motif TGGCCC which has been shown to bind the liver-specific transcription factor LF-A1.
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PMID:Transcriptional regulation of the human insulin receptor promoter. 132 34

During differentiation of 3T3-L1 preadipocytes into adipocytes, the transcription of adipocyte genes, including the stearoyl-CoA desaturase 2 (SCD2) gene, is activated. Transfection experiments with chimeric SCD2 promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs revealed a preadipocyte repressor element (PRE) capable of repressing transcription of the reporter gene in preadipocytes but not in adipocytes. DNase I protection and gel retardation analyses were used to localize the PRE site between nucleotides -435 and -410 of the SCD2 promoter and to identify a nuclear PRE binding protein present at high levels in preadipocytes and HeLa cells but lacking or inactive in adipocytes. Southwestern blot analysis indicated that the PRE binding protein has an apparent molecular mass of approximately 58 kDa. A single copy of the PRE site, inserted upstream of the simian virus 40 enhancer/promoter of pSV2CAT, was capable of strongly repressing transcription of the reporter gene in preadipocytes and HeLa cells but not in adipocytes. Taken together these results suggest that the PRE site and binding protein may regulate transcription of SCD2 and possibly other adipocyte genes by inhibiting their transcription in preadipocytes.
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PMID:Identification of a transcriptional repressor down-regulated during preadipocyte differentiation. 135 1

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

In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.
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PMID:Promoter region of the human alpha 2A adrenergic receptor gene. 138 31

The interactions of nuclear proteins from embryonal carcinoma cells (PCC3) with the long terminal repeats (LTRs) of murine intracisternal A particle (IAP) genes were studied. Two protein-DNA complexes were detected between PCC3 nuclear extract and IAP LTRs in a gel mobility shift assay. An additional complex was observed when enriched fractions from a heparin-agarose column were used as the source of proteins. Two regions within the LTR of IAP 81 were identified as the sites of protein interaction by DNase I protection. One region encompasses 43 nucleotides within the U3 region at the 5' end of LTR 81. The other covers a 78 base pair region lying within 100 nucleotides upstream from the transcription initiation site. Studies using constructs containing intact or deleted versions of the LTR fused to the bacterial chloramphenicol acetyltransferase gene indicated that the absence of the 5' 47 base pairs reduced the level of chloramphenicol acetyltransferase transcription to 20% of that driven by the entire LTR. Southwestern analysis of PCC3 nuclear extracts and column fractions revealed that a 28,000- and a 46,000-dalton protein were the major species that interact with the 5' end of IAP LTR 81.
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PMID:Nuclear protein binding to the 5' enhancer region of the intracisternal A particle long terminal repeat. 140 Apr 31

Basal expression of a chimeric gene (pMHO4CAT) consisting of approximately 7 kilobase pairs (kbp) of the 5'-flanking region of the mouse heme oxygenase-1 (HO-1) gene fused to the bacterial chloramphenicol acetyltransferase gene is 2- to 10-fold greater than that of an analogous construct containing only 1287 bp of the 5'-flanking region (pMHO1CAT) in transiently transfected cultured cells. The enhancer activity has been localized to a 268-base pair (bp) fragment positioned approximately 4 kilobase pairs upstream of the transcription initiation site. This fragment contains two high affinity protein binding sites, regions A and B, as determined by DNase I protection assays using nuclear protein extracts from rat C6 glioma cells. Both sites include core sequence elements, TGAGTCA (region A) and TGTGTCA (region B), that resemble the consensus binding site, TGA(G/C)TCA, of the Jun/Fos (AP-1) family of transcription factors. Purified, bacterially expressed AP-1 (c-Jun homodimer) specifically binds to both elements, exhibiting greater affinity for the region A motif. The expression of pMHO4CAT, but not of pMHO1CAT, is stimulated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the 268-bp enhancer fragment confers TPA inducibility and c-Jun/c-Fos transactivation to the heterologous SV40 promoter. These functions are mediated by the AP-1 binding sites as multiple copies of the region A motif also confer TPA induction and c-Jun/c-Fos transactivation upon a heterologous promoter.
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PMID:Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene. 140 Apr 99


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