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)

The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5' deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5' deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.
Biochem J 1992 Sep 01
PMID:Cloning of a functional Burkitt's lymphoma polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) gene promoter by the polymerase chain reaction, and its interaction with inducible cellular factors. 138 10

In rat, gamma-glutamyl transpeptidase (GGT) is encoded by multiple mRNAs (mRNAI, mRNAII, mRNAIII, and mRNAIV) that differ only in their 5' untranslated regions and are transcribed from a single-copy gene. Using oligonucleotides designed from the 5' untranslated sequences of the GGT mRNAII and mRNAIII, we amplified a 3.4-kb genomic sequence which contains the promoter region for mRNAII. The sequence flanking the two initiation start sites for mRNAII contains consensus motifs for several potential regulatory proteins and a TATA-like element at the expected position 26 bp upstream from the predominant start site. The sequence from positions -528 to +72 associated with the chloramphenicol acetyltransferase (CAT) reporter gene drives a promoter activity in LLC-PK1, a pig kidney cell line. Deletion analysis revealed that the region from nucleotides -528 to -322 mediates an activation of the promoter activity, whereas the sequence from -322 to -114 has a negative effect. Furthermore, the structural organization of the 5' end of the GGT gene reveals that the GGT mRNAIII is transcribed from a third promoter located upstream from the promoter II on the GGT gene. By Northern blot analysis, the promoter II was found to be expressed only in the kidney and in the epididymis. We also identified two new mRNA species which are expressed in the H5 hepatoma cells. Therefore, the GGT gene expression reveals a strong tissue- or cell-specific pattern which is based on the transcription of several mRNA species from multiple promoters.
Biochemistry 1992 Sep 29
PMID:Identification of a second promoter which drives the expression of gamma-glutamyl transpeptidase in rat kidney and epididymis. 138 88

In order to establish alternatives to the frequently used uterotropic assay with mice, defined estrogen-sensitive cell lines (MCF-7 cells and LeC-9 cells) were used to determine the estrogenic activities of purified compounds of vegetable origin (myco- and phytoestrogens) and zearalenone-contaminated forage cereals (wheat, barley and oats). In MCF-7 cells, a human breast cancer cell line, the induction of an estrogen-specific exoprotein served as a parameter of estrogenic activities. LeC-9 cells represent a genetically transformed cell clone derived from mouse L-cells. Here, hormone-like activities were measured by the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an estrogen-responsive element. Toxic effects affecting cell viability were monitored in this system by the expression of a second reporter gene (the bacterial beta-galactosidase gene controlled by the constitutive human beta-actin promoter). Relative estrogenic activities of myco- and phytoestrogens determined with both systems are concomitant, but higher as compared to the uterotropic assay with mice.
Toxicology 1992 Sep
PMID:Validation of two in vitro test systems for estrogenic activities with zearalenone, phytoestrogens and cereal extracts. 138 42

Two compatible plasmids were recently reported [Ikeda et al. (1992) Nucleic Acids Res. 20, 2517-2524] that together can be used to determine whether a mutant T7 RNA polymerase promoter is active or inactive in vivo. The first plasmid, pKGP1-1, carries T7 gene 1 (the gene encoding T7 RNA polymerase) ligated to a tac promoter, while the second plasmid, pCM-X#, carries the gene encoding chloramphenicol acetyltransferase (CAT) ligated to potential T7 promoters. If the pCM-X# plasmid carries a potential T7 promoter that can be utilized by T7 RNA polymerase, then CAT is produced from transcripts generated by T7 RNA polymerase from the potential promoter on the pCM-X# plasmid. To determine whether Escherichia coli growth characteristics and chloramphenicol (cam) resistance produced by the plasmids pKGP1-1 and pCM-X# reflect the T7 promoter activity of the possible promoters carried by the pCM-X# plasmids, the in vivo and in vitro strengths of the potential T7 promoters were compared and correlated. In vivo promoter strength was determined by measuring the relative amounts of CAT present in E. coli extracts, while relative in vitro promoter strength was measured in transcription assays. The in vivo and in vitro strengths of 22 point mutants of the consensus T7 promoter were shown to correlate with the growth characteristics and cam resistance conferred to E. coli harboring the plasmid pKGP1-1 and the respective pCM-X# plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 1992 Sep 22
PMID:In vivo and in vitro activities of point mutants of the bacteriophage T7 RNA polymerase promoter. 139 Jun 94

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.
Eur J Biochem 1992 Sep 15
PMID:Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency. 139 64

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to inflammation, its expression is increased by 1000-fold, primarily because of a 200-fold increase in the rates of SAA gene transcription. We have shown that when 304 bp of 5' flanking region of the rat SAA1 gene is fused to a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, CAT activity is induced in a cell-specific manner in response to conditioned media prepared from activated mixed lymphocyte cultures and recombinant interleukin-1. In this study, deletion of the SAA1 promoter to -120 bp with respect to the transcriptional start site did not diminish promoter activity; however, deletion to -94 bp renders the promoter completely inactive. Functional analysis have demonstrated that a 66-bp DNA fragment spanning -138 bp to -73 bp could confer cytokine responsiveness to a heterologous thymidine kinase promoter. Within this 66-bp responsive element resided an NF kappa B-like-binding site and a C/EBP-like-binding site. Although each binding site alone could confer responsiveness when stimulated with conditioned media and TPA, the response was much weaker than that observed when both sites were present. Moreover, site-specific mutations of either binding site completely abolished SAA1 promoter activity. Taken together, these results suggest a functional importance for and cooperative interaction of these two nuclear-factor binding sites in the cytokine-induced expression of the rat SAA1 gene.
Nucleic Acids Res 1992 Sep 25
PMID:Cooperative effects of C/EBP-like and NF kappa B-like binding sites on rat serum amyloid A1 gene expression in liver cells. 140 89

Carnitine palmitoyltransferase (CPT) regulates the flux of long-chain fatty acids into the mitochondria for subsequent beta-oxidation. A 485 bp segment of the promoter for the gene encoding the 68 kDa CPT was isolated from a rat lambda DASH genomic library using the polymerase chain reaction. The promoter contained a consensus binding sequence for CREB (cyclic AMP response element binding protein) at -153 to -166, and for C/EBP alpha (CCAAT/enhancer binding protein) at -115 to -128. DNAase I footprinting using proteins isolated from rat liver nuclei indicated the presence of several regions of nuclear protein binding, most notably at -95 to -130, at -273 to -295, and at a wide region encompassing -395 to -465. DNAase I footprinting studies with purified CREB and C/EBP alpha confirmed that protein binding to DNA occurred at the sites predicted by the consensus sequences. The segment containing 481 bp of 5' flanking sequence plus 181 bp of untranslated mRNA was ligated to the structural gene for chloramphenicol acetyltransferase (CAT). When this plasmid was transfected into Hep G2 cells, CAT activity was stimulated 7-fold by addition of 1 mM-8-bromo-cyclic AMP (8-Br-cAMP) or co-transfection of the expression vector coding for the catalytic subunit of protein kinase A (PKA). The ability of several known second messengers and transcription factors to stimulate transcription of 68 kDa CPT promoter-CAT reporter was tested in co-transfection experiments. 68 kDa CPT promoter-CAT reporter transcription activity was stimulated 7-fold by addition of 8-Br-cAMP, and this induction was depressed 50% by the addition of phorbol esters. When the 68 kDa CPT promoter-CAT reporter was co-transfected with an expression vector for CREB or C/EBP alpha, transcription was increased 3- and 10-fold respectively. 8-Br-cAMP caused an additional 8-fold induction in the presence of each factor to yield 25- and 80-fold induction respectively. Co-transfection of the expression vector for c-jun also increased the CAT activity driven by the 68 kDa CPT promoter, while co-transfection with the expression vector for c-fos had no effect. When expression vectors for both c-jun and c-fos were co-transfected with the 68 kDa CPT promoter, c-fos depressed the induction seen with c-jun alone.
Biochem J 1992 Sep 15
PMID:Isolation and characterization of the promoter for the gene coding for the 68 kDa carnitine palmitoyltransferase from the rat. 825 Aug 54

A series of simian virus 40-immortalized hepatocyte cell lines which are heterogeneous with regard to expression of albumin protein and RNA were characterized for their ability to transcribe the albumin gene. Nascent chain extension assay showed that albumin RNA levels in these cells were determined predominantly at the transcription level. The albumin promoter and enhancer sequences were fused to the bacterial chloramphenicol acetyltransferase gene; the ability of the resulting expression constructs to drive chloramphenicol acetyltransferase expression after transfection into these hepatocyte cell lines was measured. The activity of the albumin promoter and enhancer constructs in primary hepatocytes was also measured. The albumin promoter was expressed differentially in these cells; however, no correlation was found between the transcriptional efficiency of the transfected albumin promoter and endogenous albumin transcription. The albumin enhancer was functional in some but not all albumin-positive cells. The minimal albumin enhancer was mapped to a 330-base pair fragment extending from -9.94 kilobases (kb) to -10.27 kb; three elements within this fragment recently shown to be necessary for enhancer function in a murine hepatocyte cell line were also essential for albumin enhancer function in the rat hepatocyte cell line CWSV1. A transcriptional silencer was identified which could suppress the expression of the homologous albumin promoter and the heterologous herpes simplex virus thymidine kinase promoter. Preliminary analysis localized the albumin silencer between -11 and -12 kb. Our results suggest that multiple regulatory sequences may act cooperatively to determine efficient tissue-specific expression of the albumin gene.
Cell Growth Differ 1992 Sep
PMID:Functional analyses of albumin expression in a series of hepatocyte cell lines and in primary hepatocytes. 141 9

The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.
Protein Eng 1992 Sep
PMID:Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86). 143 64

The pregnancy-specific beta 1 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5' flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5' further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G+C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.
Mol Biol Rep 1992 Sep
PMID:Nucleotide sequence of a pregnancy-specific beta 1 glycoprotein gene family member. Identification of a functional promoter region and several putative regulatory sequences. 145 58


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