Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In view of the wide chromosomal distribution of short alternating purine-pyrimidine sequences capable of adopting a number of superhelical stress-dependent structural configurations (left-handed helices and cruciforms), the question has been posed whether such sequences exert any functional effects in vivo. A series of eukaryotic expression vectors were constructed which contained C.G tracts of various lengths in the promoter region. It was shown that insertion of C.G tracts of 12-16 bp significantly increased the level of expression of the chloramphenicol acetyltransferase reporter gene. It was also demonstrated that the formation of additional activation complexes and the use of a preferred "face" or side of the DNA molecule is not responsible for the increased transcription which was observed upon insertion of the C.G tracts. Comparative assays of chromatin structure at the chimeric promoters indicate that the alternating C.G tracts adopt a structure which is incapable of binding histone proteins. These results strongly suggest that control of access to chromatin is involved in regulating the transcriptional activity of the chimeric promoters. Possible molecular bases for this phenomena are discussed.
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PMID:Enhancement of transcription by short alternating C.G tracts incorporated within a Rous sarcoma virus-based chimeric promoter: in vivo studies. 884 44

We have explored different domains within the hepatitis C virus (HCV) 5' noncoding region as potential targets for inhibition of HCV translation by antisense oligodeoxynucleotides (ODNs). Inhibition assays were performed with two different cell-free systems, rabbit reticulocyte lysate and wheat germ extract, and three types of chemical structures for the ODNs were evaluated: natural phosphodiesters (beta-PO), alpha-anomer phosphodiesters (alpha-PO), and phosphorothioates (PS). A total of six original ODNs, displaying sequence-specific inhibition ranging from 62 to 96%, that mapped in the pyrimidine-rich tract (nucleotides [nt] 104 to 127) and in the initiator AUG codon (nt 338 to 357) were identified. Two ODNs, which were targeted at the initiatory AUG (nt 341 to 367 and 351 to 377) and which had been previously described as active against genotype 1b and 2a sequences, were shown to exhibit inhibition of expression (> 95%) of a type 1a sequence. Control experiments with the irrelevant chloramphenicol acetyltransferase sequence as a marker and randomized ODNs demonstrated that levels of inhibition associated with the use of PS compounds (of as much as 94%) were mainly due to nonspecific effects. Both alpha- and beta-PO ODNs were found equally active, and no difference could be seen in the activity of beta-PO when it was tested in either rabbit reticulocyte lysate or wheat germ extract, suggesting that RNase H-independent mechanisms may be involved in the inhibitions observed. However, specific RNA cleavage products generated from beta-PO inhibition experiments could be identified, indicating that, with these compounds, control of translation also involves RNase H-dependent mechanisms. This study further delimits the existence of favorable target sequences for the action of ODNs within the HCV 5' noncoding region and indicates the possibility of using nuclease-resistant alpha-PO compounds in cellular studies.
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PMID:In vitro inhibition of hepatitis C virus gene expression by chemically modified antisense oligodeoxynucleotides. 889 Nov 41

A purine/pyrimidine mirror repeat element (M-PMR3) in the MUC1 promoter has been shown to form H-DNA under in vitro conditions. We investigated this element for biological function in the regulation of transcription of this gene. Chloramphenicol acetyltransferase reporter-promoter constructs were prepared in which the mirror repeat element (PMR3) was intact, deleted, or modified, and their activities were evaluated by transient transfection assays into the cell lines Capan-2, PANC1, and HT-29. Deletion or modification of M-PMR3 increased expression of chloramphenicol acetyltransferase activity in MUC1-expressing cells; however, a role for an H-DNA structure in this activity was not supported by the results.
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PMID:A potential H-DNA element in the MUC1 promoter does not influence transcription. 890 Jan 24

In Trypanosoma brucei, pre-mRNAs are joined to a 5' 39 nt spliced leader sequence by trans splicing, a process that has not been well characterized. We have asked whether the 3' splice site regions of human and yeast introns are able to substitute in vivo for the 3' spliced leader acceptor regions of trypanosome pre-mRNA sequences. The ability of heterologous sequences to participate in trans splicing in trypanosomes was assayed by chloramphenicol acetyltransferase (CAT) enzyme activity and/or the detection of spliced CAT mRNA. Four out of the six heterologous 3' splice site regions (human beta-globin intervening sequence (IVS)2, human c-myc IVS2, human factor-VIII IVS1, and yeast actin IVS) functioned as 3' spliced leader acceptor regions in T. brucei, while two did not show significant or detectable levels of CAT activity (human beta-globin IVS1 and human c-myc IVS1). In the case of the human beta-globin IVS1 however, lengthening of the polypyrimidine tract as a result of single purine to pyrimidine transversions produced an active acceptor in which the spliced leader addition site coincides with the 3' splice site of the beta-globin exon 2. These studies indicate that some, but not all 3' acceptor regions in humans can function as spliced leader addition sites in trypansomes.
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PMID:Human and fungal 3' splice sites are used by Trypanosoma brucei for trans splicing. 901 Aug 38

An initiator tRNA gene, metA, and a closely linked fragment of a second initiator-tRNA-like sequence, metB, from Mycobacterium tuberculosis H37Ra have been cloned and characterized. The promoter region of metA shows the presence of conserved sequence elements, TAGCCT and TTGGCG, with resemblance to -10 and -35 promoter regions. The deduced sequence of the mature tRNA contains the three unique features of the eubacterial initiator tRNAs represented by (i) a C:U mismatch at position 1:72, (ii) three consecutive base pairs, 29-31G:C39-41 in the anticodon stem, and (iii) a purine:pyrimidine (A:U) base pair at position 11:24 in the dihydrouridine stem. A putative hairpin structure consisting of an 11 bp stem and a three-base loop found in the 3' flanking region is followed by a stretch of T residues and may serve as a transcription terminator. Analysis of the expression of metA and of its promoter using chloramphenicol acetyltransferase fusion constructs in Mycobacterium smegmatis shows that metA is a functional gene driven by a strong promoter. Furthermore, the overexpressed transcripts are fully processed and formylated in vivo. The metB clone shows the presence of sequences corresponding to those downstream of position 30 of the tRNA. However, the CCA sequence at the 3' end has been mutated to CCG. Interestingly, the 3' flanking sequences of both the genes are rich in GCT repeats. The metB locus also harbours a repeat element, IS6110. A method to prepare total RNA from mycobacteria (under acidic conditions) to analyse in vivo status of tRNAs is described.
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PMID:Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis. 938 37

Ultraviolet light induces the expression of tumor necrosis factor alpha (TNF alpha) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V increased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNF alpha expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNF alpha gene expression in mouse and human cells.
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PMID:UV-DNA damage in mouse and human cells induces the expression of tumor necrosis factor alpha. 964 8

Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in-frame AUG codons initiate the long open reading frame (positions 734-736 and 740-742). The extra-cistronic region extending between the uncapped 5'-end and the ORF contains two pyrimidine-rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140. In order to study the relative contribution of these elements to the process of internal initiation of translation, cDNA representations of the 5'-terminal extra-cistronic region of HAV RNA were inserted in the intergenic region of the bi-cistronic plasmid pSV-GH/CAT, between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT), and following transfection of COS-1 cells, the transient expression of both genes was quantified. The importance of the 3'-PRT appeared to be strongly influenced by the length of the 'spacer' sequence extending between this structure and the translation initiation site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation to occur. Bringing the length of the 'spacer' back to its actual size in HAV RNA (i.e. 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to this process of the 3'-PRT became marginal. Concomitantly, the importance of the functional domains previously identified in the 5'-PRT fluctuated: while integrity of domain 100-106 was always stringently required for initiation to occur, the activity of domain 113-118 paralleled that of the 3'-PRT, and the opposite applied to domain 121-126, whose contribution became relevant only after switching off the 3'-PRT. Systematic mutations introduced in the 'spacer' sequences suggest that the length of this region may be responsible for the down regulation of translation of HAV RNA and, possibly, for its lengthy replication cycle.
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PMID:The distance between the 3'-pyrimidine-rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA. 973 41

RNA-dependent protein kinase PKR is an important regulator of gene expression in interferon (IFN)-treated and virus-infected cells. The 50-kb gene encoding human PKR kinase (pkr) is inducible by IFN. Transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the human pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription. Sequence determination and mutational analysis of the pkr promoter region revealed, in addition to a functional copy of the IFN-stimulated response element (ISRE) responsible for inducibility by type I IFN, a novel 15-bp element required for optimal promoter activity mediated by the ISRE. This element (5' GGGAAGGCGGAGTCC 3'), designated KCS for kinase-conserved sequence, is exactly conserved between the human and mouse pkr promoters in sequence and position relative to the ISRE. We have now carried out an extensive mutational analysis of the 15-bp KCS element. Site-directed mutagenesis was performed, whereby every base pair position within the KCS element was replaced by each of the other three alternatives. Forty-five substitution mutants were analyzed for promoter activity by transient transfection analysis of untreated and IFN-treated human cells. The results establish 5' NNRRRGG(C,A,T)GGRGYYN 3', where R stands for purine and Y stands for pyrimidine, as the consensus sequence for the KCS element, both for basal and for IFN-inducible promoter activity. KCS-binding proteins were detected by electrophoretic mobility shift analysis (EMSA). Competition EMSA established that constitutively expressed nuclear proteins bound the KCS element selectively; KCS protein binding activity correlated with promoter activity in the transient transfection reporter assay.
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PMID:Mechanism of interferon action: identification of essential positions within the novel 15-base-pair KCS element required for transcriptional activation of the RNA-dependent protein kinase pkr gene. 981 30

The stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is regulated by oxygen tension in the pheochromocytoma-derived PC12 cell line. We previously identified a pyrimidine-rich 27-base-long protein-binding sequence in the 3'-untranslated region of TH mRNA that is associated with hypoxia-inducible formation of a ribonucleoprotein complex (hypoxia-inducible protein-binding site (HIPBS)). In this study, we show that HIPBS is an mRNA stabilizing element necessary for both constitutive and hypoxia-regulated stability of TH mRNA. The mutations within this sequence that abolish protein binding markedly decrease constitutive TH mRNA stability and ablate its hypoxic regulation. A short fragment of TH mRNA that contains the wild-type HIPBS confers the increased mRNA stability to the reporter chloramphenicol acetyltransferase mRNA. However, it is not sufficient to confer hypoxic regulation. The HIPBS element binds two isoforms of a 40-kDa poly(C)-binding protein (PCBP). Hypoxia induces expression of the isoform 1, PCBP1, but not the isoform 2, PCBP2, in PC12 cells.
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PMID:Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3'-untranslated region. 989 Oct 25

A genetic system for the study of ribosomal RNA function and structure was developed. First, the ribosome binding sequence of the chloramphenicol acetyltransferase gene and the message binding sequence of 16S ribosomal RNA were randomly mutated and alternative highly functional sequences were selected and characterized. From this set of mutants, a single clone was chosen and subjected to a second round of mutagenesis to optimize the specificity of the system. In the resulting system, plasmid-encoded ribosomes efficiently and exclusively translate specific mRNA containing the appropriate ribosome binding sequences. This system allows facile isolation and analysis of mutations that would normally be lethal and allows direct selection of rRNA mutants with predetermined levels of ribosome function. The system was used to examine the effects of mutations at the sole pseudouridine (Psi) in Escherichia coli 16S rRNA which is located at position 516 of the conserved 530 loop. The nucleotide opposite Psi516 in the hairpin, A535, was also mutated. The data show that a pyrimidine (Psi or C) is required at position 516, while substitutions at position 535 reduce ribosome function by < 50%. A requirement for base pair formation between Psi516 and A535 was not indicated.
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PMID:Genetic approaches to studying protein synthesis: effects of mutations at Psi516 and A535 in Escherichia coli 16S rRNA. 1169 35


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