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)

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.
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PMID:Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. 631 19

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the adult circulation, is produced by a wide range of cell and tissue types. IGFBP-3 appears to be regulated by transcriptional and/or posttranslational mechanisms in a species-, cell-, and development-specific manner. In vitro and in vivo studies suggest that a number of factors (e.g. cAMP, GH, insulin-like growth factor-I, epidermal growth factor, TSH, and FSH) can act as transcriptional regulators of IGFBP-3 in particular cell types. To address the mechanistic basis for these observations, we isolated the rat IGFBP-3 gene and began characterization and analysis of the hormonal regulation of its promoter. The rat IGFBP-3 gene is located within 2 adjacent EcoRI fragments spanning about 10 kilobases. Southern analysis indicated a single copy gene. A 1.18-kilobase fragment 5' to the translation initiation codon has been sequenced and showed 65% homology with the corresponding human IGFBP-3 sequence. The region between -100 and -1 bp relative to the transcription start site showed 85% homology. The transcription start site was 118 basepairs (bp) up-stream of the initiation codon, and a TATA box consensus was located 27 bp 5' to this CAP site. No CAAT box was present, but a CpG island was identified. Consensus sequences for a number of putative response elements (e.g. activating protein-2, insulin, TSH/insulin-like growth factor, and GH) were present within -700 bp of the CAP site. A series of 5'-truncated chloramphenicol acetyltransferase reporter constructs has been transfected into both COS-1 cells and the rat thyroid cell line FRTL-5. Both basal and hormonally responsive (TSH and phorbol ester) promoter activities have been localized within the first 472 bases of the promoter region. These data indicate that suitable transfected cell systems can be established in which additional investigations can be undertaken into the mechanisms of cell- and species-specific hormonal regulation of IGFBP-3 gene expression.
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PMID:Cloning and characterization of the promoter for the rat insulin-like growth factor-binding protein-3 gene. 753 Jun 50

HIV-1, in contrast to animal retroviruses, is not lysed by human complement, but is readily inactivated by the sera from different animal species. To identify a possible species-specific protection mechanism. HIV-1 was expressed in cells of non-human origin. Recombinant HIV-1 virions that could encode the chloramphenicol acetyltransferase (CAT) protein were produced in African green monkey COS-1 cells, mink cells and, as a control, in human HEp-2 cells and were then used to infect CD4-positive target cells. Analysis of the CAT activity of the target cells revealed that fresh HIV-1-negative human serum reduced the infectivity of HIV-1 derived from monkey and mink cells five- to tenfold, but had no effect on HIV-1 produced in human cells. In addition, human serum efficiently lysed HIV-1 produced in non-human cells in contrast to HIV-1 originating from human cells, suggesting lysis as an important mechanism of virus inactivation. Mammalian cells are protected against lysis by homologous complement by membrane-bound regulatory molecules. Two of these complement inhibitors, namely decay-accelerating factor (DAF) and, to a lesser extent, CD59 were found on the surface of HIV-1 virions by means of a virus capture assay. Antibodies against DAF, but not against other host cell molecules found on the viral surface, efficiently blocked the resistance of HIV-1 produced in human cells to human complement. These results suggest that the acquisition of DAF during the budding process from human cells protects HIV-1 in a species-specific way against the attack of human complement.
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PMID:Decay-accelerating factor (CD55) protects human immunodeficiency virus type 1 from inactivation by human complement. 753 Nov 47

Phosphorylation of HeLa SII (or TFIIS)-related nuclear protein p21/SIIR was demonstrated in transfected COS-1 cells. To test for a possible functional link between phosphorylation and the previously described Rous sarcoma virus (RSV) long terminal repeat (LTR) repression (Yeh, C.H., and Shatkin, A.J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11002-11006), p21/SIIR mutants were constructed and assayed for phosphorylation level and effect on RSV LTR-driven chloramphenicol acetyltransferase (CAT) reporter expression. A major phosphorylation target in p21/SIIR was localized to the Arg/Ser-rich region between amino acids 12 and 49. Deletion of this region impaired the ability of p21/SIIR to down-regulate RSV LTR promoter function. Four serine pairs, all displaying the Arg/Lys-Ser-Ser motif typical of phosphorylation sites, are present in p21/SIIR between positions 31 and 48. Conversion of these individual serine pairs to alanine resulted in decreased phosphorylation in each case. Mutation of the Ser36-Ser37 pair also diminished by severalfold the repression activity of p21/SIIR. The single tyrosine (Tyr155) in p21/SIIR was not detectably phosphorylated in transfected COS-1 cells, suggesting that the Ser36-Ser37 pair mediates Ser/Thr phosphorylation of p21/SIIR and is critical for LTR repression function.
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PMID:The Ser36-Ser37 pair in HeLa nuclear protein p21/SIIR mediates Ser/Thr phosphorylation and is essential for Rous sarcoma virus long terminal repeat repression. 759 88

3,5,3,'-Triiodothyroacetic acid (Triac) has been used in therapy of resistance to thyroid hormone on an empirical basis and appears beneficial in some studies. We observed that the T3 analogs, Triac and 3,5,3'-triiodothyropropionic acid (Triprop), have a higher affinity for the thyroid hormone receptor-beta 1 (TR beta 1) than does T3 (2.7- and 1.8-fold, respectively), whereas the affinities of the three compounds for TR alpha 1 are the same. To evaluate whether T3 analogs would have a differential effect on TR beta 1 and TR beta 1 mutants and thus be a specific treatment for patients with resistance to thyroid hormone, we examined the induction of the transcriptional activation of wild-type (wt) TR alpha 1, TR beta 1, and mutant TR beta 1s by T3, Triac, and Triprop. The dose response of transcriptional activation by T3 analogs was measured by transient cotransfections with TRs and a rat malic enzyme-TRE fused to thymidine kinase (TK)-chloramphenicol acetyltransferase (CAT) in COS-1 cells. For TR alpha 1 wt, induction of CAT activity by T3 and Triac occurred at the same concentration. For TR beta 1 wt, Triac and Triprop showed a higher maximal activity than T3 (Tripro > Triac > T3) and reached 50% induction at a lower concentration than T3 (Tripro < Triac < T3). Induction of CAT activity in five mutant TR beta 1s (kindreds Mh, Mc, CL, Mf, and GH) was also analyzed. Even high levels of T3 analogs could not restore CAT activity to that of TR beta 1wt for any mutant. A dominant negative effect was produced by Mh, Mc, and Mf. Mutants CL and GH had a mild dominant negative effect depending on T3 analog concentrations and TREs. Cotransfection studies were performed using a rat malic enzyme-TK-CAT reporter plasmid to analyze the effects of hormones at near-physiological concentrations of T3 and Triac. Triac had a significantly higher transcriptional activation than T3 in Mc, CL, and GH, suggesting that Triac would have a beneficial effect to different degrees for different mutant TR beta 1s. Using mutants Mc and GH, further studies were carried out using rat GH and double palindromic and inverted palindromic TREs in COS-1 cells. On each TRE, 10 nmol/L Triac induced higher transcriptional activation in TR beta 1wt, mutant TR beta 1s, and TR beta 1wt plus mutant TR beta 1s (1:1 ratio) than the same dose of T3.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Triiodothyroacetic acid has unique potential for therapy of resistance to thyroid hormone. 760 51

The 5'-terminal untranslated region (5' UTR) of the uncapped hepatitis A virus (HAV) RNA contains two pyrimidine-rich sequences; one about 20 nucleotides (nt) in length in the vicinity of the AUG initiation codon (nt 706-726), and a longer one (about 40 nt) encompassing nt 100 to 140. The latter includes a 13 nt 'core' sequence (positions 126-138 in the HM175 strain) which is 80% identical to the pyrimidine-rich tract of poliovirus type 1 RNA (Mahoney strain). Representative cDNAs of the entire 5' UTR of HAV RNA were inserted in the intercistronic region of the bi-cistronic plasmid pSV-GH/CAT between the genes coding for the human growth hormone (GH) and bacterial chloramphenicol acetyltransferase (CAT). When COS-7 cells were transfected with these constructs they transiently expressed CAT indicating that the 5' UTR of HAV was efficiently directing internal initiation of translation of the reporter gene. Under similar conditions the 5' UTR of poliovirus type 2 (Lansing strain) was 30% more efficient in directing the expression of the CAT gene. Removal of the 'core' sequence from the 5'-distal pyrimidine-rich stretch extending between nt 117 and 131 in the HAV 5' UTR reduced the CAT activity in the lysates of transfected cells by 40%, whereas point mutations engineered in this segment strongly decreased (80% inhibition) the HAV-driven expression of the reporter gene. Limited mutations systematically introduced in the reiterated (U)UUUCCC motifs of the 5'-distal pyrimidine-rich tract identified two major functional domains extending between nt 100-106 and 113-119. Substitutions in these hexanucleotides abrogated internal initiation of translation, whereas similar changes in the neighbouring domains (nt 107-112 and 120-126) had no effect on the expression of the reporter gene, suggesting that the 5'-most pyrimidine-rich tract is indeed part of the structure(s) recognized by ribosomes and associated factors at initiation of translation and that the hexanucleotides 100-106 and 113-119 constitute an important part of it. Although HAV replicates better at 33 degrees C than at 37 degrees C, incubation of transfected cultures at 33 degrees C delayed the expression and slightly reduced the level of CAT activity in the cell lysates, but the overall effect of the mutations remained unchanged.
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PMID:5' UTR of hepatitis A virus RNA: mutations in the 5'-most pyrimidine-rich tract reduce its ability to direct internal initiation of translation. 773 Aug 3

A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P) of human parainfluenza virus type 3 (HPIV-3). Two plasmids, one containing the amino terminus of P fused to the DNA-binding domain of the yeast transactivator, GAL4, and the other containing the amino terminus of NP fused to the herpesvirus transactivator, VP16, were transfected in COS-1 cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding sites. A specific and high-affinity interaction between NP and P was observed as measured by the activation of the CAT gene. Mapping of the domains in P (603 amino acids) involved in the association with NP revealed that NH2-terminal 40 and COOH-terminal 20 amino acids are important for such association. Interestingly, a stretch of NH2-terminal amino acids as short as 63-403 interacted with NP more than the wild type, reaching greater than 2.5-fold as measured by the CAT assay. These results suggest that a domain is present in P that negatively regulates its interaction with NP. Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95%. These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression.
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PMID:Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3. Mapping of the interacting domains using a two-hybrid system. 775 93

We developed a novel method for analysis of hepatitis C viral proteinase activity in cultured cells, in which the proteinase activity was measured as the enhancement of reporter gene expression. In this system, plasmids encoding a reporter gene, the enzyme gene, and the substrate gene were simultaneously transfected into COS-1 cells. The reporter plasmid contains chloramphenicol acetyltransferase (CAT) gene downstream of an enhancer/promoter sequence derived from the human T-cell leukemia virus type-1 (HTLV-I) long-terminal repeat (LTR). The substrate expression plasmid was a triple chimera; HCV nonstructural protein 2 (NS2) and the Tax1 protein of HTLV-I sandwiched the substrate polypeptide, which was inserted upstream of Tax1. This method assumes that since the HCV NS2 appears to be located in the lipid bilayer of endoplasmic reticulum (ER) membranes, the Tax1 of the chimeric substrate was trapped on the surface of the ER in the absence of HCV proteinase activity. After release from the chimera by HCV proteinase-dependent cleavage, Tax1 could transactivate the expression of the CAT gene through the enhancer sequence of HTLV-I LTR. This system should enable us to simply and safely screen the potential antiviral activity of proteinase inhibitors in vivo, although this system may be limited to proteinase inhibitors that are permeable to the plasma membrane.
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PMID:A novel method for analysis of viral proteinase activity encoded by hepatitis C virus in cultured cells. 777 61

HeLa cell basic nuclear protein (p21), which represses Rous sarcoma virus long terminal repeat (RSV LTR) promoter activity, diminished v-src expression and the appearance at permissive temperature of the transformed phenotype in tsRSVLA23 Rat-1, a cell line transformed with a temperature-sensitive mutant of RSV. Nuclear run-on analyses using COS-1 cells cotransfected with p21 cDNA and chloramphenicol acetyltransferase reporter indicated that p21 inhibits transcription initiation by targeting a region in the RSV LTR promoter between positions -108 and -85 upstream of the cap site. Insertion of this 24-base pair sequence in place of one of the 72-base pair enhancers in the SV40 early promoter rendered it sensitive to p21 repression. Electrophoretic mobility shift assays using a synthetic oligomer corresponding to the 24-base pair LTR promoter element revealed that p21 altered the pattern of protein.DNA complex formation apparently without binding DNA directly. Complex formation assayed by UV cross-linking and DNA affinity chromatography indicated further that a cellular factor which can interact with this element was decreased in cells transfected with p21 expression plasmid. The results indicate that p21 repression of RSV LTR is mediated by a cis-acting element and may occur by alteration of protein complexes formed on this promoter element.
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PMID:A cis-acting element in Rous sarcoma virus long terminal repeat required for promoter repression by HeLa nuclear protein p21. 779 84

Among human sarcomas, osteosarcomas usually display high intrinsic mdr1 expression while malignant fibrous histiocytomas (MFH) do not. A comparative polymerase chain reaction (PCR)-based sequence analysis of the mdr1 promoter revealed point mutations in seven out of nine osteosarcomas at nucleotides +103 (2 cases T-->C) and +137 (5 cases G-->T). No changes were seen in eight MFHs. When COS cells transfected with CAT constructs containing the T-->C chloramphenicol acetyltransferase mutant mdr1 promoters were treated with vincristine or doxorubicin, expression of the CAT gene was enhanced to a higher extent than with constructs containing wild-type or G-->T-mutant mdr1 promoters. We suggest that there is a correlation between the type of mdr1 promoter mutation and responsiveness to MDR relevant drugs.
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PMID:Point mutations in the mdr1 promoter of human osteosarcomas are associated with in vitro responsiveness to multidrug resistance relevant drugs. 783 15


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