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)

Sporamin, the tuberous root storage protein of the sweet potato, accounts for about 60 to 80% of the total soluble protein of this organ. The amount of sporamin present in other organs is very low, or even not detectable, in the normal field-grown plants. However, the stem of sweet potato plantlets grown axenically on agar medium containing sucrose was found to accumulate large amounts of sporamin. Two-dimensional gel electrophoretic profiles of sporamin precursors synthesized in vitro by poly(A)+ RNA are indistinguishable between tuberous roots of the field-grown plants and stems of the axenically cultured plants, suggesting that an essentially identical set of the members of sporamin multigene family are expressed in these two organs under different growth conditions. Transgenic tobacco plants having a CAT (chloramphenicol acetyltransferase) fusion gene with the 5' upstream region of a sporamin A gene, gSPO-A1, show preferential expression of CAT activity in stems when the plants are maintained in axenic culture on sucrose medium as is the case for sporamin in sweet potato. Deletion analysis revealed that the DNA sequence of gSPO-A1 between -94 and -305, relative to the transcription start site, is important for its expression in tobacco. This region contains two of the previously postulated putative regulatory elements conserved between sporamin A and B genes.
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PMID:High-level expression of tuberous root storage protein genes of sweet potato in stems of plantlets grown in vitro on sucrose medium. 210 38

The stimulatory effects of the 13S adenovirus E1A gene product on the human cytomegalovirus (HCMV) major immediate early (IE) enhancer were examined. Chimeric plasmids containing cloned portions of the HCMV major IE enhancer-promoter positioned upstream of the chloramphenicol acetyltransferase gene (cat) were cotransfected into HeLa cells with the plasmid p13S-wt which contained a cDNA encoding the adenovirus 13S E1A gene product. CAT expression from chimeric plasmids containing at least one copy of the HCMV 19 base pair (bp) repetitive motif was stimulated 10-fold in the presence of p13S-wt. The 19-bp motif contains a potential binding site for the cellular transcription factor ATF/CREB. Deletion analysis indicated that the ATF/CREB site was crucial for E1A-mediated stimulation. Insertion of a synthetic oligonucleotide homologous to a 19-bp motif and containing an ATF/CREB binding site into an HCMV chimera lacking ATF/CREB motifs conferred E1A responsivity on HCMV promoter-mediated CAT expression whereas insertion of a similar oligonucleotide containing a change of two bases in the sequence of the ATF/CREB site did not. Measurement of CAT-specific RNA verified the results of the CAT enzyme experiments. The ATF/CREB motif may be a target for stimulation of HCMV gene expression through either viral or cellular transcription factors.
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PMID:The human cytomegalovirus immediate early enhancer-promoter is responsive to activation by the adenovirus-5 13S E1A gene. 214 16

Phytochrome negatively regulates the transcription of its own phyA genes. High levels of Pfr, the active, far-red-light absorbing form of phytochrome, repress phyA transcription; low Pfr levels result in derepression. We have utilized microprojectile-mediated gene transfer to identify regions of an oat phyA3 gene involved in this autoregulation. Chimeric constructs containing various deletion and sequence substitution mutants of the oat phyA3 gene fused to a chloramphenicol acetyltransferase reporter (phyA3/CAT) have been introduced into etiolated rice seedlings by particle bombardment. Low Pfr concentrations induce high phyA3/CAT expression, whereas high Pfr represses activity to near basal levels. Removal of phyA3 sequences 3' to the transcription start site reduces expression about fivefold, suggesting that intron 1 of the phyA3 gene may be required for high activity. The degree of high-Pfr-imposed repression is unaffected by any of a series of deletions or sequence substitutions in the phyA3 promoter, thus providing no evidence of any Pfr-activated negative elements. In contrast, 5' and internal deletions identify a minimum of three major positive promoter elements, designated PE1 [-381 base pairs (bp) to -348 bp], PE2 (-635 bp to -489 bp), and PE3 (-110 bp to -76 bp) that are necessary for high-level expression in low-Pfr cells. The data indicate that PE1 and PE2 are functionally redundant, but that PE3 is required in conjunction with either PE1 or PE2 for activity. PE3 contains a sequence element that is highly conserved between monocot phyA promoters, indicative of a critical role in phyA expression.
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PMID:cis-acting elements involved in photoregulation of an oat phytochrome promoter in rice. 215 9

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.
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PMID:Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells. 216 40

An iso-1-cytochrome c-chloramphenicol acetyltransferase fusion protein (iso-1/CAT) was expressed in Saccharomyces cerevisiae and used to delineate two stages in the cytochrome c import pathway in vivo (S. H. Nye and R. C. Scarpulla, Mol. Cell. Biol. 10:5753-5762, 1990 [this issue]). Fusion proteins with the CAT reporter domain in its native conformation were arrested at the initial stage of mitochondrial membrane recognition and insertion. In contrast, those with a deletional disruption of the CAT moiety were relieved of this block and allowed to translocate to the intermembrane space, where they functioned in respiratory electron transfer. In the present study, iso-1/CAT was used to map structural determinants in apoiso-1-cytochrome c involved in the initial step of targeting to the mitochondrial membrane. Carboxy-terminal deletions revealed that one of these determinants consisted of the amino-terminal 68 residues. Deletion mutations either within or at the ends of this determinant destroyed mitochondrial targeting activity, suggesting that functionally important information spans the length of this fragment. Disruption of an alpha-helix near the amino terminus by a helix-breaking proline substitution for leucine 14 also eliminated the targeting activity of the 1 to 68 determinant, suggesting a contribution from this structure. A second, functionally independent targeting determinant was found in the carboxy half of the apoprotein between residues 68 and 85. This determinant coincided with a stretch of 11 residues that are invariant in nearly 100 eucaryotic cytochromes c. Therefore, in lieu of an amino-terminal presequence, apocytochrome c has redundant structural information located in both the amino and carboxy halves of the molecule that can function independently to specify mitochondrial targeting and membrane insertion in vivo.
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PMID:Mitochondrial targeting of yeast apoiso-1-cytochrome c is mediated through functionally independent structural domains. 217 85

H4IIE rat hepatoma cells were stably transfected with various phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase (PEPCK-CAT) expression vectors. The regulation of the transfected genes was qualitatively similar to that of the endogenous PEPCK gene. CAT expression was increased in response to cAMP and dexamethasone and insulin overrode these effects at concentrations known to be effective in suppressing transcription of the endogenous gene. The effect of insulin was dominant, as it is with the endogenous gene. A series of 5',3', and internal deletions of the PEPCK gene promoter were used to show that this insulin response requires at least two separate elements. One insulin-responsive sequence is located between -468 and -402, relative to the transcription initiation site. The other is between -271 and +69.
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PMID:Regulation of phosphoenolpyruvate carboxykinase gene expression by insulin. Use of the stable transfection approach to locate an insulin responsive sequence. 217 98

Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.
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PMID:Expression of recombinant androgen receptor in cultured mammalian cells. 217 2

We compared the ability of HIV-1 tat protein and JCV T-antigen in inducing transcription from the JCV late promoter, JCVL. A JCVL promoter-chloramphenicol acetyltransferase plasmid (pJCL-CAT) was transfected into human glial cells alone or together with plasmids producing T-antigen and tat protein. CAT enzyme activity obtained from the transfected cells indicated that both JCV T-antigen and HIV-1 tat proteins stimulated JCV late gene expression. However, the level of induction mediated by tat protein was significantly higher than that obtained with T-antigen. Moreover, in contrast to JCV T-antigen, tat stimulated JCVL-promoter activity over a narrow range of ptat expressor plasmid concentration. Co-transfection of both T-antigen and tat plasmids at optimal concentrations resulted in greater than additive CAT activity from the JCVL promoter. This synergism suggests that the two activator proteins utilize alternative mechanisms to exert their effects. Using deletion mutations from the 5' end of the JCVL promoter, we demonstrated that different regions within the JCV enhancer/promoter are important for T-antigen and tat induction, implying that these activators function through distinct targets to increase JCVL promoter activity.
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PMID:Regulation of the human neurotropic virus promoter by JCV-T antigen and HIV-1 tat protein. 217 36

High level bacterial resistance to chloramphenicol is generally due to O-acetylation of the antibiotic in a reaction catalysed by chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) in which acetyl-coenzyme A is the acyl donor. The crystal structure of the type III enzyme from Escherichia coli with chloramphenicol bound has been determined and refined at 1.75 A resolution, using a restrained parameter reciprocal space least squares procedure. The refined model, which includes chloramphenicol, 204 solvent molecules and two cobalt ions has a crystallographic R-factor of 18.3% for 27,300 reflections between 6 and 1.75 A resolution. The root-mean-square deviation in bond lengths from ideal values is 0.02 A. The cobalt ions play a crucial role in stabilizing the packing of the molecule in the crystal lattice. CAT is a trimer of identical subunits (monomer Mr 25,000) and the trimeric structure is stabilized by a number of hydrogen bonds, some of which result in the extension of a beta-sheet across the subunit interface. Chloramphenicol binds in a deep pocket located at the boundary between adjacent subunits of the trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically essential histidine belongs to the adjacent subunit. His195 is appropriately positioned to act as a general base catalyst in the reaction, and the required tautomeric stabilization is provided by an unusual interaction with a main-chain carbonyl oxygen.
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PMID:Refined crystal structure of type III chloramphenicol acetyltransferase at 1.75 A resolution. 218 98

A synthetic gene coding for the winter flounder antifreeze protein (AFP) has been constructed. A new strategy for the synthesis has been employed such that one strand of the duplex was chemically synthesized and the other was produced enzymatically by chain extension. The chemically synthesized blocks were constructed so that the second strand was self-priming. The resulting DNA fragment was incorporated into the vector, pGCS1, which contained a translational fusion of the sequence encoding AFP and the N terminus of cat (encoding chloramphenicol acetyltransferase, CAT), under the control of the cauliflower mosaic virus 35S promoter. This plasmid was introduced into protoplasts of corn (var. Black Mexican Sweet) by electroporation. Production of the fusion peptide was monitored by CAT assay and Western blotting with antisera to AFP and CAT.
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PMID:Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells. 221 Mar 78


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