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

Human papillomavirus type 8 (HPV8) belongs to the HPV types associated with skin carcinomas of patients with epidermodysplasia verruciformis (EV). Its noncoding regulatory sequences (NCR) were shown to drive the expression of the reporter gene chloramphenicol acetyltransferase (cat) in transient assays with human epithelial cells (HT3 cells). This constitutive activity could be enhanced by coexpression of the HPV8 transactivator protein E2. The analysis of 5' deletions of the NCR showed that the EV-specific sequence motif M33 and the neighboring AP1 site are essential for the promoter activity, whereas 44 nucleotides located immediately upstream of M33 are strongly inhibitory. The same effects were observed in simian virus 40-immortalized fetal keratinocytes (SV61 cells) and spontaneously immortalized skin keratinocytes (HaCaT cells). By using primer extension and RNase protection analyses two promoters could be identified within the HPV8 NCR. A nested set of weak signals, corresponding to start sites between positions 175 to 179, represented the previously described E6 promoter. The vast majority of transcripts was initiated at position 7535 and shown to undergo processing at an NCR-internal splice donor (positions 1 to 8). The promoter P7535 is similar to late promoters of other skin-associated papillomaviruses as far as localization, transcript structure, and sequence characteristics are concerned. To confirm that P7535-initiated transcripts proceed indeed to the L1 gene for the major capsid protein, viral mRNAs from an HPV8-induced lesion of a patient with EV were characterized by RNase protection and sequence analysis of polymerase chain reaction-amplified cDNAs. The NCR leader (positions 7535 to 4) appeared in two messages with three exons each. The third exon started with the second ATG codon of L1 in both cases; the short central exons from the 3' part of the early coding region were defined by a common splice acceptor site (position 3303) and different splice donor sites (positions 3443 and 3704).
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PMID:Late promoter of human papillomavirus type 8 and its regulation. 131 64

A plasmid carrying the 5'-flanking region (-1584 to +47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5- to 9-fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of E1A resides in a common region(s) of 13S and 12S E1A products. The major target region of E1A was mapped within the 68 base-pair region (-21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2F, although the upstream region (-83 to -21) including ATF(CREB)-binding consensus had an additional effect in the transactivation.
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PMID:Activation of the mouse proliferating cell nuclear antigen gene promoter by adenovirus type 12 E1A proteins. 135 54

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

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.
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PMID:Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells. 152 81

A plasmid carrying the 5'-flanking region (-1852 to +33 with respect to the transcription initiation site) of the mouse DNA polymerase beta gene fused with the chloramphenicol acetyltransferase (CAT) gene was cotransfected into mouse N18TG2 cells with adenovirus type 12 E1 genes-expressing plasmids. Expression of E1A gene products resulted in the elevation of the CAT expression by 3 to 7 folds, but that of E1B gene product was much less effective. RNase protection analysis revealed that the activation by E1A was at the transcription process. Both the 13S E1A and the 12S E1A activated the DNA polymerase beta gene promoter, indicating that the activation domain of E1A is in a common region(s) of 13S and 12S E1A products. The major target sequence of E1A was mapped within the 10 base pair-region (-30 to -20) of the DNA polymerase beta gene promoter, which overlapped with the palindromic sequence known as the ATF(CREB)/E4F-binding consensus. The results suggest that the palindromic sequence is essential for E1A-induced transcriptional activation of the mouse DNA polymerase beta gene.
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PMID:Activation of the mouse DNA polymerase beta gene promoter by adenovirus type 12 E1A proteins. 153 5

Human selenium-dependent glutathione peroxidase (hGPx1) (EC 1.11.1.9) is thought to be involved in many critical cellular functions as a result of its role in glutathione-mediated reduction of toxic peroxides, and it is implicated as a mechanism of resistance against oxygen free radicals. Previous studies have demonstrated that the gene encoding hGPx1 (hgpx1) is more highly expressed in multidrug-resistant AdrR MCF-7 human breast cancer cells than in the parental WT MCF-7 cell line. In order to further study the transcriptional regulation of hgpx1, we have cloned the genomic hgpx1 gene and determined its nucleotide sequence. The 2550-base pair (bp) 5'-flanking sequence of hgpx1 contained the terminal 511 bp of the 3' end of a previously reported rhoH12 cDNA (Yeramian, P., Chardin, P., Madaule, P., and Tavitian, A. (1987) Nucleic Acids Res. 15, 1989), a ras-related oncogene. Further downstream from rhoH12, but before the start of transcription of hgpx1, RNase protection analysis revealed a transcribed sequence of at least 270 bp which we have called mid. RNA transcripts homologous to both rhoH12 (1.8 and 1.5 kilobase pairs (kb)) and mid (1.8 kb) are also more highly expressed in AdrR MCF-7 cells than in WT MCF-7 cells. We screened an AdrR MCF-7 cDNA library with the mid sequence and isolated a partial cDNA clone which contains both mid and rhoH12 sequences and is colinear with the genomic sequence which extends from 10 bp 3' to the rhoH12 stop codon to 810 bp 5' to the start of transcription of hgpx1. The start of transcription of hgpx1 in AdrR MCF-7 cells was determined by primer extension analysis. The promoter and 2 kb of the 5'-flanking sequence of hgpx1 was fused to the bacterial chloramphenicol acetyltransferase gene (hGPx1-CAT1). Analysis of deletion constructs of hGPx1-CAT1 revealed three possible cis-acting regulatory regions. The transcriptional regulation of hgpx1 was examined using the hGPx1-CAT hybrid genes and nuclear run-on studies. We found no evidence that increased mRNA transcript formation could account for different levels of hgpx1 RNA either in different breast cancer cell lines or in response to selenium.
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PMID:Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1). 155 8

We have cloned and characterized 5'-flanking sequences of the DNA methyltransferase (MeTase) gene. DNA MeTase gene transcription is initiated at a few discrete sites: 343 and 90 base pairs upstream of the translation initiation site as determined by RNase protection and primer extension assays. The promoter sequences that regulate expression of DNA MeTase, as defined by chloramphenicol acetyltransferase assays, reside between position -171 and the transcription start site. The promoter of DNA MeTase does not contain TATAA or CAAT boxes and is unusual because it does not contain the CG-rich elements characteristic of TATAA-less housekeeping genes. The 5'-flanking region of DNA MeTase contains AP-1, AP-2 and glucocorticoid response elements, suggesting possible regulation by cellular signal transduction pathways. The base composition of the DNA MeTase promoter is markedly different from that of other housekeeping genes. Whereas most housekeeping genes are characterized by CG-rich areas in their 5'-flanking regions, the TG dinucleotide is over-represented in DNA MeTase 5'-flanking sequences, including a perfect tandem repeat of T/G between positions -685 and -650. DNA methylation patterns play an important role in the developmental regulation of gene expression in vertebrates. DNA MeTase activity is probably regulated to maintain this pattern of methylation. We suggest that the DNA MeTase promoter represents a new class of housekeeping gene promoters that was designed to ensure high fidelity regulation of gene expression.
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PMID:The mouse DNA methyltransferase 5'-region. A unique housekeeping gene promoter. 155 80

The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5'-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5'-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.
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PMID:Isolation and characterization of the human sucrase-isomaltase gene and demonstration of intestine-specific transcriptional elements. 156 17

The int-2 gene, which encodes a member of the fibroblast growth factor family, is expressed at specific sites and times during mouse development. In certain embryonal carcinoma cell lines, multiple int-2 transcripts accumulate when the cells are induced to differentiate with retinoic acid and dibutyryl cyclic AMP. Nuclear run-on analyses indicate that the apparent induction of int-2 expression results from an increase in the rate of transcription initiation. Six distinct types of RNA have been delineated, originating from three promoters and terminating at either of two polyadenylation sites. Since each transcript appears to encode the same protein, this complexity may reflect the need for lineage-specific or differentiation-dependent control of expression. By comparing the kinetics of induction and turnover of the different RNA species, we show that the choice of promoter or length of the 3'-untranslated region has no significant effect on the half-lives of the various mRNAs. To further evaluate control at the transcriptional level, we have shown that a 1.7-kilobase fragment of int-2 genomic DNA, when fused to the chloramphenicol acetyltransferase gene, can act as a regulated promoter(s) in differentiated versus undifferentiated embryonal carcinoma cells. This segment of DNA encompasses the three promoter regions previously delineated by RNase mapping plus about 900 base pairs of additional upstream sequences.
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PMID:Transcriptional regulation of the int-2 gene in embryonal carcinoma cells. 164 13


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