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)

At least two genes encode isoenzymes of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Alternative splicing of one of these genes generates a skeletal muscle-specific transcript from an upstream promoter and a liver-specific transcript from a downstream promoter. A potent glucocorticoid response element was identified in the first intron of the gene, i.e. between liver exon I and exon II. The element is approximately 3.5 kilobase pairs (kb) downstream of the liver isoenzyme transcription start site and 13 kb upstream of exon II of the gene and confers dexamethasone-sensitive expression of chloramphenicol acetyltransferase (CAT) activity from a heterologous thymidine kinase promoter and from both homologous 5'-flanking regions of the gene. This glucocorticoid response element also exhibits androgen- but not estrogen-sensitive expression of CAT activity in HeLa cells cotransfected with the appropriate receptor expression vector. DNase footprint and sequence analysis revealed that the element is comprised minimally of two adjacent 15-mer glucocorticoid receptor dimer binding sites situated in opposite orientations. Glucocortcoid regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in liver and skeletal muscle is mediated by a single complex glucocorticoid response element located in the first intron of the skeletal muscle/liver gene.
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PMID:Regulation of gene expression of rat skeletal muscle/liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Isolation and characterization of a glucocorticoid response element in the first intron of the gene. 133 34

The activity of the human O6-methylguanine-DNA methyltransferase (MGMT) gene promoter was determined in eight human cell lines by measuring chloramphenicol acetyltransferase activity in a reporter gene system. MGMT promoter activities in cells that do not express MGMT (Mer-) fell within the range of activities seen in cells that do express MGMT (Mer+). The promoter region contains 11 potential binding sites for the transcription factor Sp1, but no correlation was seen between cellular Sp1 protein and MGMT promoter chloramphenicol acetyltransferase activity. Because Mer- cells are not deficient in the factors needed for transcription of MGMT, we suggest that at least two mechanisms regulate MGMT expression. One suppresses MGMT mRNA and protein in Mer- cells, and another regulates the levels of constitutive expression in Mer+ cells. Sp1 is not a limiting factor in MGMT expression.
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PMID:A comparison of human O6-methylguanine-DNA methyltransferase promoter activity in Mer+ and Mer- cells. 142 89

Antisense oligodeoxynucleotide (ODN), which are directed against the splice acceptor site of exon II of the regulatory gene tat of the human immunodeficiency virus type 1 (HIV-1), have been described. These 20-mer ODN's displayed moderate anti-HIV activity in vitro. Using the same antisense ODN (termed ODN-2), which was additionally modified and protected both at the 3'- and the 5'-terminus by two phosphorothioate internucleotide linkages, a strong anti-HIV activity (EC50: 2.7 micrograms/ml) could be measured in the HIV-1/CEM- and HIV-1/HeLa-T4+ cell system. The analogous ODNs which were protected only at one end were either inactive (up to 10 micrograms/ml) or displayed a low antiviral activity. Time kinetic studies revealed that the antisense ODN-2 reduced the release of HIV-1 already after an incubation time of 1 h. By applying S1 nuclease protection procedures, it could be established that the antisense ODN-2 inhibited splicing of high molecular weight transcript to the 2-kb tat mRNA in HIV-1-infected CEM cells. Transfection experiments with pU3R-III chloramphenicol acetyltransferase expression vector in HeLa-T4+ cells revealed that the antisense ODN-2 blocked the Tat protein-mediated transactivation process. In co-transfection experiments using pSV2tat72 or scrape loading studies with purified Tat, the transactivation was restored. These data indicate that the selected antisense ODN-2 displays its anti-HIV effect by blocking the splicing process leading to the functional 2-kb tat mRNA.
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PMID:Antisense oligodeoxynucleotide: inhibitor of splicing of mRNA of human immunodeficiency virus. 156 36

cAMP-dependent protein kinase appears to play a role in cAMP-induced gene expression in mammalian cells. There exist two major types of cAMP-dependent protein kinase, type I and type II, which are distinguished by their regulatory subunits, RI and RII, respectively. We investigated the role of type I and type II protein kinase in the cAMP-induced gene expression by either stable or co-transfection of RI alpha, RII alpha, or RII beta gene in an expression vector together with somatostatin-chloramphenicol acetyltransferase (SS-CAT) fusion gene using a cAMP-unresponsive mutant pheochromocytoma cell line (A126-1B2). Introduction of the RII beta gene restored the capability of these cells to induce the SS-CAT gene expression in response to forskolin stimulus and induced a changed morphology which resembled that of wild type. The RII alpha gene also induced SS-CAT gene expression but to a lesser degree than that achieved by the RII beta gene, whereas the RI alpha gene had no effect. The induction of SS-CAT gene expression by the RII beta gene was specifically blocked by the 21-mer RII beta antisense oligodeoxynucleotide. These results show for the first time that type II but not type I regulatory subunit of cAMP-dependent protein kinase is essential for a cAMP-induced gene transcription.
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PMID:Type II regulatory subunit of protein kinase restores cAMP-dependent transcription in a cAMP-unresponsive cell line. 197 35

Translational coupling was demonstrated in a gene fusion in which the promoter and the N-terminal region of the Bacillus subtilis subtilisin (aprA) gene were fused to a promoterless Tn9-derived chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. Expression of this gene fusion results in the production of a native-sized CAT product, whereas the Tn9-derived CAT gene is usually not translated from its own ribosome binding site in B. subtilis (D. S. Goldfarb, R. L. Rodriguez, and R. H. Doi, Proc. Natl. Acad. Sci. USA 79:5886-5890, 1982). A 178-base-pair deletion, which removed part of the signal peptide and the propeptide of the aprA gene and created a translational stop codon 230 base pairs upstream of the CAT gene ribosome binding site, reduced expression of the CAT gene. A BamHI 10-mer linker insertion into this deletion site, which restored the reading frame and simultaneously removed the translation stop codon, restored CAT gene expression. The data indicate that expression of the CAT gene was dependent on translation of the truncated aprA gene into the ribosome binding site of the CAT gene.
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PMID:Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion. 299 17

Two recombinant clones, lambda LC5 and lambda LC13, encompassing the entire regulatory myosin light chain 2 (MLC2A) gene of chicken heart muscle were isolated. Of these, lambda LC5 which contains a large 5'-flanking sequence of about 7.0 kb, was characterized by a partial nucleotide sequence analysis. A TATA-like sequence (TATTTTTA) and a CAAT-box (CAAAAGT) are located at positions -32 and -59, respectively, which most likely constitute the functional promoter region in the gene. Based on primer extension reaction with a synthetic 20-mer corresponding to the 5'-leader sequence and total poly(A+) RNA, the probable transcription initiation site in the gene was located. The gene promoter activity was demonstrated following transient expression of recombinant genomes containing the chicken upstream sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) or to the rat preproinsulin II genes. The extracts from a Quail fibroblast cell line (QT35) transfected with the construct (pLCo5.2iCat) containing the putative chicken promoter, and the CAT gene promoted the formation of 3'-acetate chloramphenicol. Another construct (pBC12LC5.2f) contains the rat preproinsulin II gene placed under the control of chicken promoter and a simian virus 40 origin of replication. Transfection of COS cell line with pBC12LC5.2f DNA resulted in an efficient expression of rat preproinsulin mRNA initiating from the chicken promoter. The transfection assay also allowed detection of chicken MLC2A gene transcripts by S1-nuclease protection of end-labeled DNA probes. A comparison of the MLC2A upstream gene sequence with those available for skeletal myosin light chains revealed no common sequence elements, suggesting that cardiac MLC2A gene promoter region has diverged considerably from its counterparts in skeletal muscle.
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PMID:Characterization of 5'-flanking region of heart myosin light chain 2A gene. Structural and functional evidence for promoter activity. 302 54

O6-Methylguanine-DNA methyltransferase plays an important role in cellular defence against mutagens and carcinogens with alkylating activity. Certain tumor-derived cell lines, termed Mer-, are defective in the enzyme activity and have an increased sensitivity to alkylating agents. We cloned the genomic sequence coding for the human O6-methylguanine-DNA methyltransferase and elucidated the structure. The gene consisted of 5 exons and spanned more than 170 kb, while mRNA for the enzyme was 950 nucleotides long. No or only little mRNA for the enzyme was formed in Mer- cells, though there was no gross difference in the coding and promoter regions of the gene between Mer+ and Mer- cells. The putative promoter region, derived from Mer+ cells, was placed upstream of the chloramphenicol acetyltransferase reporter gene and the constructs were introduced into Mer+ and Mer- cells. In Mer- cells, a lowered level of transient expression of the gene was observed as compared with Mer+ cells, but this difference alone does not account for the in vivo difference of expression of the gene in the two types of cells; there might be difference in cis-acting elements. The DNA sequence in the 5' upstream region of the gene was extremely GC-rich and there were no consensus sequences, such as the TATA and CAAT boxes. There were lower levels of methylation in the putative promoter of various Mer- cells, as compared with findings in Mer+ cells. Methylation in this region may be involved in regulating expression of the gene.
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PMID:Organization and expression of the human gene for O6-methylguanine-DNA methyltransferase. 767 40

Transcription of both Xenopus thyroid hormone receptor (TR) genes, xTR alpha and -beta, is strongly up-regulated by their own ligand T3 during natural or T3-induced metamorphosis of tadpoles and in some Xenopus cell lines. To explain this autoinduction, we analyzed the sequence of 1.6 kilobases of xTR beta promoter for putative T3-responsive elements. Two direct repeat +4 AGGTCA hexamer motifs (DR+4), an imperfect distal (-793/-778) and a perfect proximal (-5/11) site, a DR+1 site, and some possible half-sites were located in the 1.6-kilobase promoter. Transfection of Xenopus XTC-2 cells (which express xTR alpha and -beta) and XL-2 cells (which predominantly express TR alpha) with chloramphenicol acetyltransferase reporter constructs of deletion mutants and promoter fragments showed that the distal and proximal DR+4 sites responded to T3, although other flanking sequences may also play a role. The thyroid hormone-responsive element half-site present as DR+1 in the up-stream sequence at -1260/-950, when cloned in front of a heterologous promoter, functions independently. T3 enhanced transcription from the two DR+4-containing fragments when present together by only 2- to 3-fold due to a high basal activity. Overexpression of unliganded xTR alpha and xTR beta in XTC-2 cells repressed basal activity, which was then enhanced 7- to 4-fold by T3, respectively; with XL-2 cells cotransfected with xTR beta, T3 inducibility increased to 16-fold. Electrophoretic mobility shift assays with recombinant Xenopus TR alpha, TR beta, retinoid-X receptor-alpha (RXR alpha) and RXR gamma proteins showed that TR-RXR heterodimers, but not TR or RXR monomers or homodimers, strongly bound the natural and synthetic distal and proximal DR+4 elements in a ligand-independent manner. TR/RXR heterodimers exhibited the highest binding affinity for a 28-mer oligonucleotide probe for the -5/11 proximal DR+4 site, with only slight binding to DR+1 (retinoid-X-responsive element-like) site. The xTR beta promoter binding to XTC-2 cell nuclear extract suggested the in vivo relevance of the findings with recombinant TR/RXR heterodimers. It is concluded that xTR alpha and -beta proteins are capable of regulating the expression of xTR beta gene, which can explain its autoinduction seen during T3-induced metamorphosis.
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PMID:Analysis of structure and expression of the Xenopus thyroid hormone receptor-beta gene to explain its autoinduction. 776 Aug 54

Type 1 plasminogen activator inhibitor (PAI-1) is the major physiological inhibitor of plasminogen activation, inhibiting both tissue- and urokinase-type plasminogen activators. In HTC rat hepatoma cells, glucocorticoids increase PAI-1 activity, antigen and mRNA accumulation 3- to 5-fold; this increase is due solely to an increase in the rate of PAI-1 gene transcription. We have identified the cis-acting sequences in the 5'-flanking sequence of the HTC PAI-1 gene that mediate this induction. Analysis of a series of hybrid genes containing various portions of the PAI-1 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene transfected into HTC cells localized the region involved in the transcriptional regulation by glucocorticoids to between -1237 and -764. Electrophoretic mobility shift assays and DNase-I protection assays showed that a glucocorticoid response element (GRE) 15-mer located at -1212 bound the glucocorticoid receptor DNA-binding domain protein in a concentration-dependent manner. Mutations created within this GRE eliminated its ability both to confer a glucocorticoid response and to bind the glucocorticoid receptor. When placed upstream of a heterologous promoter in either orientation, this GRE conferred glucocorticoid inducibility. We, therefore, conclude that the sole cis-acting sequence required for the glucocorticoid response of the PAI-1 gene in rat HTC hepatoma cells is the GRE at -1212.
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PMID:Mechanism of glucocorticoid induction of the rat plasminogen activator inhibitor-1 gene in HTC rat hepatoma cells: identification of cis-acting regulatory elements. 824 19

Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal structure of the catalytic domain, i.e. residues 382 to 637, of Azotobacter vinelandii E2p (E2pCD) was solved by multiple isomorphous replacement and refined by energy minimization procedures. The final model contains 2182 protein atoms and 37 ordered water molecules. The R-factor is 18.7% for 10,344 reflections between 10.0 and 2.6 A resolution. The root-mean-square shift deviation from the ideal values is 0.017 A for bond lengths and 3.3 degrees for bond angles. The N-terminal residues 382 to 394 are disordered and not visible in the electron density map, otherwise all residues have well-defined density. The catalytic domain forms an oligomer of 24 subunits, having octahedral 432 symmetry. In the E2pCD crystals, the 24 subunits are related by the crystallographic symmetry. The cubic arrangement of subunits gives rise to a large hollow cube with edges of 120 A. The faces of the cube have pores of diameter of 30 A. The true building block of the cube is the E2p trimer, eight of which occupy the corners of the cube. Two levels of intermolecular contacts can be distinguished: (1) the extensive interactions between 3-fold related subunits leading to a tightly associated trimer; and (2) the interactions along the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer. Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT) and comprises a central beta-sheet surrounded by five alpha-helices. The comparison of the two proteins indicates a large rotation of the N-terminal residues 395 to 426 of E2pCD, which reshapes the substrate binding site and extends the interaction between threefold related subunits. The catalytic centre consists of a 30 A long channel extending from the "inner" side of the trimer to the "outer" side, where inner and outer refer to the position in the 24-meric cubic core of the pyruvate dehydrogenase complex and correspond with CoA and lipoamide binding sites, respectively. The active site is formed by the residues with the lowest mobility as indicated by the atomic B-factors. Five proline residues surround the active site.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Refined crystal structure of the catalytic domain of dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii at 2.6 A resolution. 848


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