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)

Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-beta 1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3'-untranslated region of R2 mRNA and did not bind to the 5'UTR, the coding region of the R2 message or to the 3'UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-beta 1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3'-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-beta 1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-beta 1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-beta 1 mediated stabilization of R2 message involves a specific interaction of a p75-trans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.
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PMID:A novel transforming growth factor-beta 1 responsive cytoplasmic trans-acting factor binds selectively to the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability. 823 29

Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUGLab) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGLab for wild-type and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of eIF-2 was measured over a broad range of mRNA concentrations. In conclusion, eIF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.
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PMID:Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA. 862 30

The bcl-2 gene becomes activated in many types of human cancers and contributes to neoplastic cell expansion, as well as to resistance to radiation and chemotherapy, by blocking programmed cell death or apoptosis. The expression of this proto-oncogene is regulated at both the transcriptional and post-transcriptional levels. DNA sequence comparisons of human, mouse, rat and chicken bcl-2 cDNAs revealed the presence of an open reading frame (ORF) [correction of (OFR)] located upstream of the normal coding region. Because upstream ORFs (uORFs) have been associated with translational repression, we analysed the functional significance of the 11 amino-acid uORF in the human BCL-2 gene (-119 to -84 bp). Deletion of this uORF from chloramphenicol acetyltransferase (CAT) reporter gene constructs that contained the bcl-2 promoter and entire 5'-untranslated region (5'-UTR), as well as introduction of an A-->T mutation at position -119 bp that destroyed the AUG-initiation codon, significantly increased CAT activity in HeLa, CEM, and other cell lines, without producing a corresponding elevation in CAT mRNA levels. Positioning this uORF, together with its accompanying Kozak sequences, between a heterologous promoter from SV40 and a CAT reporter gene resulted in marked inhibition of CAT protein production without a decrease in CAT mRNA. Mutation of the start codon (ATG-->TTG) of this uORF completely abolished its inhibitory activity, consistent with a translational mechanism. Taken together, these findings suggest that the uORF located within the 5'UTR of the bcl-2 gene is necessary and sufficient for translational regulation of bcl-2 gene expression.
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PMID:A cis-acting element in the BCL-2 gene controls expression through translational mechanisms. 864 41

The receptor for hyaluronan mediated motility (RHAMM) gene expression is markedly elevated in fibrosarcomas exposed to transforming growth factor-beta1 (TGF-beta1). The half-life of RHAMM mRNA was increased by 3 fold in cells treated with TGF-beta1, indicating that growth factor regulation of RHAMM gene expression at least in part involves a posttranscriptional mechanism. Our studies demonstrated that a unique 30-nucleotide (nt) region that has three copies of the sequence, GCUUGC, was the TGF-beta1-responsive region in the 3'-untranslated region (3'-UTR) that mediated message stability. This region interacted specifically with cytoplasmic trans-factors to form multiple protein complexes of approximately 175, 97, 63, 26, and 17 kDa post-TGF-beta1 treatment, suggesting a role for these complexes in the mechanism of action of TGF-beta1-induced message stabilization. Insertion of the 3'-UTR into the chloramphenicol acetyltransferase gene conferred TGF-beta1 induced stability of chloramphenicol acetyltransferase-hybrid RNA in stably transfected cells, while the same insert carrying a deletion containing the 30-nt region had no significant effect on mRNA stability. These results provide a model of RHAMM message regulation in which TGF-beta1-mediated alteration of RHAMM message stability involves the up-regulation of multiple protein interactions with a 30-nt cis-element stability determinant in the 3'-UTR. This model also suggests that this 30-nt base region functions in cis to destabilize RHAMM mRNA in resting normal cells.
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PMID:Transforming growth factor-beta1 stimulates multiple protein interactions at a unique cis-element in the 3'-untranslated region of the hyaluronan receptor RHAMM mRNA. 866

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3'-untranslated region (3'-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3'-UTR contains regulatory information for R2 mRNA turnover. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 9-nucleotide cis-element, 5'-UCGUGUGCU-3', which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G --> A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.
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PMID:Defining a novel cis-element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA. cis-trans-interactions and message stability. 870 35

The 3'-end of mouse hepatitis virus (MHV) genomic RNA contains a recognition sequence (55 nucleotides [nt]) required for minus-strand RNA synthesis. To determine whether the 3'-end sequence is also involved in subgenomic mRNA transcription, we have constructed MHV defective interfering (DI) RNAs which contain a chloramphenicol acetyltransferase (CAT) gene placed behind an intergenic sequence and a 3'-end sequence with various degrees of internal deletions. The DI RNAs were transfected into MHV-infected cells, and CAT activities, which represent subgenomic mRNA transcription from the intergenic site, were determined. The results demonstrated that the deletions of sequence upstream of the 350 nt at the 3'-end, which include the 3'-untranslated region (3'-UTR), of MHV genomic RNA did not affect subgenomic mRNA transcription. However, deletions that reduced the 3'-end sequences to 270 nt or less completely abolished the mRNA transcription despite the fact that all of these clones synthesized minus-strand RNAs. These results indicated that mRNA transcription from an intergenic site in the MHV DI RNA requires most of the 3'-UTR as a cis-acting signal, which likely exerts its effects during plus-strand RNA synthesis. A substitution of the corresponding bovine coronavirus sequence for the MHV sequence within nt 270 to 305 from the 3'-end abrogated the CAT gene expression, suggesting a very rigid sequence requirement in this region. The deletion of a putative pseudoknot structure within the 3'-UTR also abolished the CAT gene expression. These findings suggest that the 3'-UTR may interact with the other RNA regulatory elements to regulate mRNA transcription.
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PMID:The 3' untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. 879 74

Since the short intron in the 5'-untranslated region (5'-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5'-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5'-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins11VS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides -87 and +110 showed a reduction of expression by 30%. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.
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PMID:Dual function of the intron of the rat insulin I gene in regulation of gene expression. 889 3

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.
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PMID:Subgenomic replicons of the flavivirus Kunjin: construction and applications. 899 75

The 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5'UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5'UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5'UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem-loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem-loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings, in vitro translation inhibition assays using RNA fragments of the 5'UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these two cis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5'UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. These cis-acting translational elements may serve as potential targets for the antiviral action of oligomers.
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PMID:In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5' untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers. 902 10

We have previously identified a sequence in the 3'-untranslated region (3'-UTR) of erythropoietin (Epo) mRNA which binds a protein(s), erythropoietin mRNA-binding protein (ERBP). A mutant lacking the ERBP binding site (EpoM) was generated. Hep3B cells were stably transfected with a wild-type Epo (EpoWT) cDNA or EpoM cDNA construct located downstream of a promoter of cytomegalovirus. Following inhibition of transcription, the half-lives of EpoWT and EpoM mRNAs were 7 h and 2.5 h in normoxia, respectively. The EpoM mRNA half-life remained unchanged in hypoxia. EpoWT mRNA half-life increased approximately 40% in response to a 6-h hypoxic pre-exposure and an additional approximately 50% when pre-exposed to 12 h hypoxia. The steady-state level of EpoWT mRNA was 4-fold that of EpoM mRNA reflecting the difference in mRNA decay rates in normoxia. The Epo protein level expressed from exogenous EpoM was unchanged in both normoxia and hypoxia. In contrast, the Epo protein level expressed from exogenous EpoWT increased 50% in hypoxia when compared with normoxia. These observations were further supported by chimeric chloramphenicol acetyltransferase and Epo-3'-UTR constructs. We have demonstrated that Epo mRNA stability was modulated in normoxia and further by hypoxia, therefore, providing evidence that Epo is regulated at the post-transcriptional level through ERBP complex formation.
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PMID:Post-transcriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein. 907 94


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