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)

The human genome contains a variety of elements similar in structure to retroviruses and retrotransposons. We have shown that the long terminal repeat (LTR) sequences of a large family of human retrovirus-like elements, RTVL-H, are heterogeneous in their ability to regulate the expression of linked genes. Although all of five LTRs tested could promote expression of the chloramphenicol acetyltransferase (CAT) gene, their relative promoter activities as well as range of activities varied widely. Several of the LTRs tested also exhibited bidirectional promoter activity either alone or when activated by an SV40 early enhancer. One LTR, H6, displayed strong promoter activity in human (NTera2D1, 293, Hep2), monkey (COS-1), and mouse (3T3) cells. In fact, the activity of this LTR was similar to that of the SV40 early promoter/enhancer in 293, COS-1, and 3T3 cells. RNA mapping studies have localized the transcription start site to the expected location in the H6 LTR. RTVL-H LTRs were also shown to contain sequences which could increase transcription from the human beta-globin promoter and be influenced by SV40 enhancer sequences. As the human genome contains several hundred related RTVL-H sequences and a similar number of solitary LTRs, these findings raise the possibility that RTVL-H LTRs could have diverse effects on the expression of adjacent cellular genes.
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PMID:Functional heterogeneity of a large family of human LTR-like promoters and enhancers. 169 Aug 75

We describe two transfectable vectors designed to facilitate the functional analysis of eukaryotic promoter/enhancer sequences. The first, pJFCAT1, is an improved chloramphenicol acetyltransferase (CAT) reporter gene expression vector with two features that distinguish it from the majority of other CAT vectors currently in use: 1) it carries a trimer cassette of the simian virus 40 major late polyadenylation site to block plasmid-initiated read-through expression of CAT, and 2) it includes the phage f1 origin of replication, permitting generation of single-stranded copies to serve as templates for oligonucleotide-directed mutagenesis or single-strand DNA sequencing. The promoterless pJFCAT1 directs little if any CAT activity in transfected mouse L cells and, therefore, may be particularly useful for the analysis of weak promoters whose activity is otherwise masked by background CAT expression. The second vector, pTAG-1, uses human beta-globin as a reporter gene and was designed to facilitate the analysis of reporter gene expression at the RNA level. Like pJFCAT1, pTAG-1 also includes the simian virus 40 polyadenylation site trimer cassette located just upstream of the promoter insertion site. We have used each of these vectors to study functional elements in the human and mouse thymidine kinase promoters.
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PMID:Improved expression vectors for eukaryotic promoter/enhancer studies. 172 8

Transactivator proteins of the bovine leukemia (BLV) and human T-lymphotropic (HTLV) viruses increase long terminal repeat (LTR)-directed viral gene expression and act as immortalizing oncogenes in tissue culture. We report here that the BLV tax gene sequence contains an enhancer-like activity. The X long open reading frame was cloned up-stream of the beta-globin promoter linked to the chloramphenicol acetyltransferase (CAT) gene. In the presence of tax sequences, up to sevenfold enhancement of CAT expression was observed. A computer-assisted homology search revealed the presence of a consensus enhancer core sequence (GTGTTGTTGGTTG) into the third exon of the 2.1-kb X mRNA. These studies demonstrate that the tax gene contains a transcriptional enhancer which could be involved in early viral gene expression in vivo.
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PMID:The bovine leukemia virus tax gene contains an enhancer sequence. 185 Aug 98

Artificial bicistronic mRNAs based on rabbit beta-globin and bacterial chloramphenicol acetyltransferase protein-coding sequences were tested for translation activity in a mouse astrocytoma cell-free extract. This cell extract exhibited an apparent preference for 5'-distal or internal initiation over 5'-proximal ("first AUG") initiation. 5'-Distal initiation appeared to be 5'-cap independent, suggesting that nonstandard initiation was responsible. This conclusion was based on a lack of inhibition of internal initiation by added cap analog and insensitivity of internal initiation to the presence or absence of a 5'-cap structure. Exogenous reticulocyte initiation factors were tested for effect on 5'-proximal initiation. The only factor with a significant effect was found to be eukaryotic initiation factor 4F, or the cap-binding protein. Addition of this factor promoted 5'-end initiation as evident by a general increase in 5'-proximal open reading frame (ORF) product relative to 5'-distal ORF product. The relative expression of 5'-proximal to 5'-distal ORFs in bicistronic or multicistronic mRNAs may very well be dependent on activity levels of eukaryotic initiation factor 4F and possibly other mRNA-dependent initiation factors.
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PMID:Effect of eukaryotic initiation factor 4F on AUG selection in a bicistronic mRNA. 199 20

As a first step toward elucidating the biochemical basis of gene regulation at the G1-S boundary of the cell cycle, we have identified regions of the murine thymidine kinase (TK) promoter sufficient to confer appropriately growth-responsive expression to a heterologous gene. Using a series of TK promoter-chloramphenicol acetyltransferase (CAT) gene fusion constructs, we have identified sequences located between -174 base pairs upstream and +159 base pairs downstream of the TK translation initiation site that are sufficient to drive efficient S phase-specific expression of the CAT reporter gene in transfected murine fibroblasts. Both deletion analysis and site-specific mutagenesis experiments indicated that an Sp1 consensus binding site is critical to the activity of this promoter. Synchronized populations of BALB/c 3T3 cells stably transfected with either TK promoter-CAT fusion constructs or TK promoter-beta-globin fusion constructs expressed their respective reporter genes in an S phase-specific manner following serum stimulation. In each case, reporter gene expression was reduced during quiescence and G1 and rose upon entry of cells into S phase. The TK sequences included in these constructs therefore contained information sufficient to confer S phase-specific regulation to these two reporter genes. These results set the stage for a more detailed analysis of the sequences and trans-acting factors responsible for regulating murine TK gene expression and may lead to insights into the control of proliferation in normal and transformed cells.
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PMID:Growth-responsive expression from the murine thymidine kinase promoter: genetic analysis of DNA sequences. 206 70

By utilizing chimeric genes constructed from 5'-flanking sequences of the human CYP21B (P-450C21) gene and reporter genes (chloramphenicol acetyltransferase or rabbit beta-globin), a 34-nucleotide sequence has been found to be required for cAMP-dependent transcription. This sequence (-129/-96 base pairs) shows no homology to that of the consensus (CRE) cAMP-regulatory element. Gel retardation analysis shows that a protein-DNA complex is formed between this DNA sequence and nuclear proteins from mouse adrenal Y1 tumor cells or bovine adrenal cortical cells or human fetal adrenal tissue and that formation of this complex cannot be competed by DNA containing the consensus CRE sequence. Even though cAMP-enhanced accumulation of P-450C21 mRNA in primary cultures of bovine adrenocortical cells is inhibited by the protein synthesis inhibitor, cycloheximide, reporter gene transcription enhanced by the cAMP-responsive -129/-96-base pair fragment of the human CYP21B gene is not. We conclude that cAMP-dependent transcription of the human P-450C21 gene (CYP21B), an event required for maintenance of optimal steroidogenic capacity in the adrenal cortex, involves a stable transcription factor(s) distinct from the CRE-binding protein. Furthermore the cAMP-dependent cis-regulatory element of the human P-450C21 gene is distinct from those found associated with the other steroid hydroxylase genes, 17 alpha-hydroxylase cytochrome P-450, cholesterol side chain cleavage cytochrome P-450, and 11 beta-hydroxylase cytochrome P-450, suggesting that each of these genes may require its own set of specific transcription factors for cAMP-dependent regulation.
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PMID:cAMP-dependent transcription of the human CYP21B (P-450C21) gene requires a cis-regulatory element distinct from the consensus cAMP-regulatory element. 216 43

The effect of adenovirus VA RNAI on the translation of mRNAs expressing the bacterial chloramphenicol acetyltransferase (CAT) enzyme was studied by a transient expression assay in 293 cells. The CAT activity was determined in extracts prepared from cells transfected with mixtures of plasmids encoding CAT and VA RNA. The results showed that VA RNAI co-transfection resulted in a significant increase in CAT expression from a variety of constructs. Thus, expression of CAT from a SV40 mRNA, a beta-globin mRNA and a chimeric mRNA containing the adenovirus-2 tripartite leader were all stimulated approximately 6-fold by VA RNAI. Based on these results we conclude that the tripartite leader sequence is not required for the VA RNA-mediated stimulation of translation. Our results indicate instead that VA RNAI probably functions as a general enhancer of mRNA translation. A2- to 3-fold stimulation of CAT expression was also obtained following transient expression of HeLa and CV-1 cells. The reduced efficiency was correlated with a 10- to 20-fold lower level of VA RNA expression in HeLa compared with 293 cells. Thus, it is likely that a product from region E1 indirectly enhances the translational efficiency by stimulating VA RNA transcription.
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PMID:Adenovirus VA RNAI mediates a translational stimulation which is not restricted to the viral mRNAs. 241 Feb 62

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.
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PMID:Activation of the human epsilon- and beta-globin promoters by SV40 T antigen. 252 24

Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP) affect mRNA stability. Cell extracts used in the decay reactions were depleted of functional PABP either by adding excess poly(A) competitor or by passing the extracts over a poly(A)-Sepharose column. Polyadenylated mRNAs for beta-globin, chloramphenicol acetyltransferase, and simian virus 40 virion proteins were degraded 3 to 10 times faster in reactions lacking PABP than in those containing excess PABP. The addition of purified Saccharomyces cerevisiae or human cytoplasmic PABP to PABP-depleted reactions stabilized the polyadenylated mRNAs. In contrast, the decay rates of nonpolyadenylated mRNAs were unaffected by PABP, indicating that both the poly(A) and its binding protein were required for maintaining mRNA stability. A nonspecific single-stranded binding protein from Escherichia coli did not restore stability to polyadenylated mRNA, and the stabilizing effect of PABP was inhibited by anti-PABP antibody. The poly(A) tract was the first mRNA segment to be degraded in PABP-depleted reactions, confirming that the poly(A)-PABP complex was protecting the 3' region from nucleolytic attack. These results indicate that an important function of poly(A), in conjunction with its binding protein, is to protect polyadenylated mRNAs from indiscriminate destruction by cellular nucleases. A model is proposed to explain how the stability of an mRNA could be affected by the stability of its poly(A)-PABP complex.
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PMID:The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. 256 32

Sequence analyses and dot-blot analyses with synthetic oligonucleotide probes have identified eight individuals in three Turkish families and one Bulgarian family with one chromosome having a C----T mutation at nucleotide position--101 relative to the Cap site of the beta-globin gene. This nucleotide is part of one of the conserved blocks of nucleotides within the promoter region; in vitro expression analyses with the chloramphenicol acetyltransferase system showed that this substitution will decrease the effectiveness of transcription. Five subjects had a thalassemia intermedia due to the additional presence of a known classical high hemoglobin (Hb) A2 beta-thalassemia mutation on the second chromosome; their hematologic condition was relatively mild. The three persons with a heterozygosity for the--101 C----T mutation had normal hematologic data without microcytosis but with high-normal levels of Hb A2 and a mild imbalance in chain synthesis. The newly discovered mutation is considered one of the silent types of beta-thalassemia. It is relatively rare because it was absent among several hundred normal and beta-thalassemia chromosomes.
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PMID:A C----T substitution at nt--101 in a conserved DNA sequence of the promotor region of the beta-globin gene is associated with "silent" beta-thalassemia. 271 3


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