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 compared the efficiency of human immunodeficiency virus (HIV-1) vectors that express a marker gene (chloramphenicol acetyltransferase, CAT) using different promoter elements. In one vector, CAT was expressed under the control of an internal murine leukemia virus (MuLV) long terminal repeat (LTR). In other vectors, CAT production was regulated by the HIV-1 LTR; these vectors also contained the HIV-1 tat gene and pol sequences reported to exert cis-acting positive effects on reverse transcription or gene expression. Vectors employing the Tat-driven HIV-1 LTR exhibited up to 500-fold greater CAT expression in Jurkat lymphocytes or human peripheral blood mononuclear cells compared with vectors using the internal MuLV LTR element as a promoter. This difference was not due to improved packaging of the vector RNA into virions, but to an improved level of gene expression in the target cells. Target cell CAT expression was two- to threefold higher for the vector containing the pol sequences and was only slightly less than that seen for a trans-complemented envdeleted provirus. These results indicate that defective HIV-1 vectors with efficiencies of gene transfer and expression comparable with that of HIV-1 itself are feasible.
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PMID:Use of cis- and trans-acting viral regulatory sequences to improve expression of human immunodeficiency virus vectors in human lymphocytes. 880 25

Several systems for the detection of HIV-1 have been described in which HIV-1-susceptible cells contain a reporter gene (chloramphenicol acetyltransferase, beta-galactosidase, or alkaline phosphatase) under the control of the HIV-1 long terminal repeat (LTR). Upon infection by HIV-1, the expression of the viral tat product increases transcription from the HIV-1 LTR promoter, leading to high-level expression of the reporter gene product. Previously described reporter systems require processing of the cells by lysis, fixation, or other steps following infection to detect the reporter gene product. In the present study, the Aequorea green fluorescent protein S65T variant (GFP-S65T) was used in a reporter system for detecting HIV-1. HeLa-CD4 cells transfected with the plasmid pRH1, which encodes GFP-S65T under the control of the HIV-1 LTR promoter, and either co-transfected with a plasmid encoding the HIV-1 tat product or superinfected with HIV-1, expressed high levels of GFP-S65T, which was readily detected by fluorescence microscopy and fluorescence-activated cell-sorting analysis. The advantages of this system include its simplicity, sensitivity, and ability to detect and sort live HIV-1-infected cells using readily available instruments. The construction of cell lines stably transfected with pRH1 will provide a tool for titering HIV-1 and sorting HIV-1-infected cells.
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PMID:Detection of HIV-1 infection with a green fluorescent protein reporter system. 894 67

The regulatory Tat protein of the human immunodeficiency virus type-1 (HIV-1) is essential for viral replication and also shows pleiotropic activities on various cell functions. To get further insights into the molecular mechanisms underlying the biological activity of Tat, we investigated the effect of endogenous and exogenous Tat protein on c-fos gene expression in T lymphoblastoid (Jurkat) and monocytic (U937) cell lines, as well as in primary peripheral blood mononuclear cells (PBMC). Transient cotransfection of tat cDNA in sense orientation (tat/S), together with a plasmid containing the c-fos promoter (FC3, from -711 to +42) in front of the bacterial chloramphenicol acetyltransferase (CAT) gene significantly enhanced CAT activity in Jurkat cells activated by the addition of 15% fetal calf serum (FCS) or 5 micrograms/mL phytohemagglutinin plus 10(-7) mol/L phorbol myristate acetate (PMA) and U937 cells activated by 15% FCS or 10(-7) mol/L PMA. This effect was specifically due to Tat, since Jurkat and U937 cells cotransfected either with tat cDNA in antisense orientation (tat/AS), tat carrying a mutation in the aminoacid cys22-gly22 (tat 22/S) or with the backbone vector alone (pRPneo-SL3) did not show any significant difference in c-fos promoter activity as compared to cells transfected with FC3 plasmid alone. By using deletion mutants of the c-fos promoter, we found that the minimal DNA sequence required for Tat activity was located between nucleotides -404/-220 and that the serum responsive element (SRE, -317/-288), present within this region, was still responsive to Tat. A single point mutation in the SRE completely abrogated the responsiveness to tat/S. Exogenous recombinant Tat protein was also able to upregulate c-fos promoter activity in serum-activated Jurkat and U937 cells, as well as endogenous c-fos mRNA expression and c-Fos protein synthesis in both serum-activated cell lines and primary PBMC. c-Fos protein was shown essential for an optimal transactivation of the HIV-1 long terminal repeat (LTR) by Tat: incubation of Jurkat cells with antisense, but not sense, c-fos oligonucleotides significantly reduced either the Tat-enhanced expression of an LTR-CAT reporter construct or the levels of gag p24 in the culture supernatants of Jurkat cells and PBMC acutely infected with HIV-1. Our data suggest that the c-fos upregulation mediated by Tat might play a significant role in the control of viral gene transactivation.
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PMID:Upregulation of c-Fos in activated T lymphoid and monocytic cells by human immunodeficiency virus-1 Tat protein. 905 48

In previous studies, little attention has been paid to maintaining the native HIV-1 leader sequence in reporter constructs analyzing the human immunodeficiency virus type 1 (HIV-1) promoter activity. To investigate a possible influence of the leader sequence on HIV-1-driven gene expression in the presence as well as in the absence of Tat, an expression vector was designed for transcripts consisting of the native HIV-1 tat 1.4 mRNA leader followed by the open reading frame for the bacterial chloramphenicol acetyltransferase (CAT). Deletion mutants with mutations within the leader sequence downstream of U5 (lsdU5) were constructed, as well as a mutant containing a mutation with a reverse orientation of this region. Quantification of CAT protein in HeLa-T4+ cells transiently transfected with wild-type and mutant leader constructs showed that the exon 1-derived lsdU5 region has an influence on basal as well as Tat-induced protein expression. The dramatic decrease in the level of CAT protein upon deletion of lsdU5 was paralleled by a drop in the steady-state level of CAT mRNA. Deletion of the exon 1-derived lsdU5 region also decreased the expression of mRNAs containing authentic HIV-1 sequences instead of CAT. The effect observed with the reporter constructs was not due to the loss of binding sites for nuclear factors, as could be shown with DBF1 and Sp1 mutant constructs. Nuclear run-on transcription assays showed that the presence or absence of lsdU5 did not influence the rate of transcription. This indicates that the exon 1 lsdU5 element functions at the posttranscriptional level in the processing, nucleocytoplasmic export, or stabilization of HIV-1 transcripts.
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PMID:Exon 1 leader sequences downstream of U5 are important for efficient human immunodeficiency virus type 1 gene expression. 906 Jun 29

We have approached the development of a human immunodeficiency virus type 1 (HIV-1) therapeutic product by producing immune cells stably resistant to HIV-1. Promonocytic CD4+ cells (U937) were made resistant to HIV-1 by the introduction of a DNA construct (pNDU1A,B,C) that contained three independent antisense sequences directed against two functional regions, transactivation response and tat/rev, of the HIV-1 target. Each sequence was incorporated into the transcribed region of a U1 snRNA gene to generate U1/HIV antisense RNA. Stably transfected cells expressed all three U1/HIV antisense transcripts, and these transcripts accumulated in the nucleus. These cells were subjected to two successive challenges with HIV-1 (BAL strain). The surviving cells showed normal growth characteristics and have retained their CD4+ phenotype. In situ hybridization assays showed that essentially all of the surviving cells produced U1/HIV antisense RNA. No detectable p24 antigen was observed, no syncytium formation was observed, and PCR-amplified HIV gag sequences were not detected. Rechallenge with HIV-1 (IIIB strain) similarly yielded no infection at a relatively high multiplicity of infection. As a further demonstration that the antisense RNA directed against HIV-1 was functioning in these transfected immune cells, Tat-activated expression of chloramphenicol acetyltransferase was shown to be specifically inhibited in cells expressing Tat and transactivation response region antisense sequences.
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PMID:Stable human immunodeficiency virus type 1 (HIV-1) resistance in transformed CD4+ monocytic cells treated with multitargeting HIV-1 antisense sequences incorporated into U1 snRNA. 909 86

Jembrana disease virus (JDV) is a newly identified bovine lentivirus that is closely related to the bovine immunodeficiency virus (BIV). JDV contains a tat gene, encoded by two exons, which has potent transactivation activity. Cotransfection of the JDV tat expression plasmid with the JDV promoter chloramphenicol acetyltransferase (CAT) construct pJDV-U3R resulted in a substantial increase in the level of CAT mRNA transcribed from the JDV long terminal repeat (LTR) and a dramatic increase in the CAT protein level. Deletion analysis of the LTR sequences showed that sequences spanning nucleotides -68 to +53, including the TATA box and the predicted first stem-loop structure of the predicted Tat response element (TAR), were required for efficient transactivation. The results, derived from site-directed mutagenesis experiments, suggested that the base pairing in the stem of the first stem-loop structure in the TAR region was important for JDV Tat-mediated transactivation; in contrast, nucleotide substitutions in the loop region of JDV TAR had less effect. For the JDV LTR, upstream sequences, from nucleotide -196 and beyond, as well as the predicted secondary structures in the R region, may have a negative effect on basal JDV promoter activity. Deletion of these regions resulted in a four- to fivefold increase in basal expression. The JDV Tat is also a potent transactivator of other animal and primate lentivirus promoters. It transactivated BIV and human immunodeficiency virus type 1 (HIV-1) LTRs to levels similar to those with their homologous Tat proteins. In contrast, HIV-1 Tat has minimal effects on JDV LTR expression, whereas BIV Tat moderately transactivated the JDV LTR. Our study suggests that JDV may use a mechanism of transactivation similar but not identical to those of other animal and primate lentiviruses.
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PMID:Characterization of the Jembrana disease virus tat gene and the cis- and trans-regulatory elements in its long terminal repeats. 984 71

Endogenous sulphated polysaccharides such as heparin have been shown to inhibit the infectivity of HIV-1 min vitro. However, these naturally occurring polymers, due to extensive microheterogeneity within their structure, are difficult to characterise accurately. In contrast, dextrin can be chemically sulphated to produce a series of compounds sulphated in the 2-, 3-, or 6- position, or in all 3 positions, and the use of these compounds provides an opportunity to investigate the anti-HIV-1 activity of sulphated polysaccharides. The mechanisms whereby sulphated polysaccharides exert their anti-HIV-1 activity have not been fully elucidated. The interaction of recombinant HIV-1 proteins with sulphated polysaccharides was investigated using a biotinylated derivative of dextrin 2-sulphate (D2S) in a solid phase binding system. D2S was found to bind strongly to HIV-1 tat (EC50 = 0.10 microg/mL), less strongly to CD4 (EC50 = 0.33 microg/mL), weakly to HIV-1 vif and gp160, and not at all to HIV-1 gp120 or p24. Other sulphated derivatives of dextrin, i.e. dextrin 3-sulphate, dextrin 6-sulphate and dextrin 2,3,6-trisulphate, as well as heparin and dextran sulphate, were also shown to bind to HIV-1 tat, whereas the unsulphated compound dextrin did not. Binding studies using a series of overlapping peptides representing the complete sequence of HIV-1 tat revealed that D2S bound most strongly to the core domain of HIV-1 tat, although there was also binding to the cysteine-rich domain; both of these regions are important for HIV-1 tat function. In assessing function, HIV-1 tat-mediated transactivation was measured using H938 cells, a cell line that contains the HIV-LTR (long terminal repeat) promoter linked to a chloramphenicol acetyltransferase gene. D2S significantly inhibited HIV-1 tat transactivation in a dose-dependent manner (IC50 = 0.5 microg/mL), whereas dextrin had no effect. The interaction between D2S and HIV-1 tat provides a potential mechanism of HIV-1 inhibition whereby tat is sequestered and its transactivating activity abolished, effectively inhibiting the replication cycle.
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PMID:Interaction of the transactivating protein HIV-1 tat with sulphated polysaccharides. 1007 83

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10(-9) M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit CAT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat. Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease (p < 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven CAT expression, while TNF-alpha-enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat/TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.
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PMID:Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression. 1038 Nov 71

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication is highly dependent on and modulated by interactions between viral and host cellular factors. Tat protein, encoded by one of the HIV-1 regulatory genes, tat, is essential for HIV-1 gene expression. A number of host cellular factors have been shown to interact with Tat in this process. During our attempts to determine the molecular mechanisms of Tat interaction with brain cells, we isolated a cDNA clone that encodes a novel Tat-interacting protein of 110 kDa or Tip110 from a human fetal brain cDNA library. GenBank BLAST search revealed that Tip110 was almost identical to a previously cloned KIAA0156 gene with unknown functions. In vivo binding of Tip110 with Tat was confirmed by immunoprecipitation and Western blotting, in combination with mutagenesis. The yeast three-hybrid RNA-protein interaction assay indicated no direct interaction of Tip110 with Tat transactivating response element RNA. Nevertheless, Tip110 strongly synergized with Tat on Tat-mediated chloramphenicol acetyltransferase reporter gene expression and HIV-1 virus production, whereas down-modulation of constitutive Tip110 expression inhibited HIV-1 virus production. Northern blot analysis showed that Tip110 mRNA was expressed in a variety of human tissues and cells. Moreover, digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus, and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9, two critical components for Tat transactivation function on HIV-1 long terminal repeat promoter. Taken together, these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction, and suggest that Tip110 is an important cellular factor for HIV-1 gene expression and viral replication.
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PMID:HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110. 1195 60

HIV-1 tat gene function and immunogenicity of HIV-1 Tat protein from 3 low (PS01, PS40, PS58) and 3 high (PS19, PS65, LP22) viral load infected, untreated and asymptomatic individuals from Thailand were compared. Levels of Tat-dependent chloramphenicol acetyltransferase (CAT) induced in HL3T1 cells with tat1 gene from HIV-1 isolates of high viral load group was significantly higher than those from low viral load group. HIV-1 subtype determination using env (C2-V4) gene demonstrated that 2/3 (PS01 and PS40) and 1/3 (PS58) from low viral load group were CRF01_AE and subtype B, while all 3 HIV-1 isolates from high viral load group were CRF01_AE. However, all 3 HIV-1 tat nucleotide sequences from low viral load group, which contained env CRF01_AE sequence, belonged to subtype B whereas all those from high viral load group contained CRF01_AE sequence. HIV Tat recombinant proteins from these groups were tested for immunogenicity in mice. All recombinant Tat proteins (except from PS58) were immunogenic in a dose-dependent manner, but with significantly differences of the immunogenicity levels between high and low viral load groups. These results indicated that HIV-1 subtype B tat gene activities might be associated with reduced disease progression of HIV-1 CRF01_AE infected individuals.
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PMID:HIV-1 subtype B Tat gene activities and disease progression in HIV-1 CRF01_AE infection. 1984 9


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