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)

A cellular assay is described in which transient high-level expression of a heterologous reporter gene (chloramphenicol acetyltransferase, CAT) driven by the HIV LTR is used to determine trans-activation in a cell line constitutively expressing Tat. The use of a parallel ELISA system to determine effects on expression of CAT and of the neomycin phosphotransferase (NPT) marker gene effectively eliminated sample variability caused by cumulative processing errors or cell culture conditions. In addition the use of cationic liposome-mediated transfection minimized delay between DNA treatment that initiates trans-activation and addition of inhibitors, thereby eliminating background expression levels in treated samples. The assay has the potential to discriminate between inhibition of trans-activation and nonspecific effects such as inhibition of transfection and cytotoxicity. It has been adapted to a 96-well format suitable for high-throughput screening of natural products and synthetic chemicals.
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PMID:A quantitative assay for trans-activation by HIV-1 Tat, using liposome-mediated DNA uptake and a parallel ELISA system. 825 35

We have previously shown that a human mammotropic polypeptide hormone, prolactin (PRL) can act synergistically with steroid hormones to regulate gene expression directed by the long terminal repeat of mouse mammary tumor virus (MMTV LTR) in a human ductal carcinoma cell line T47D cells using a chloramphenicol acetyltransferase reporter gene system and gene transfection methods. In the present study, using various recombinant plasmids we analyzed functional elements in the MMTV LTR that is essential for the PRL responses. We show that the PRL-responsive elements are located in the extreme 5' end of the MMTV LTR, a region previously described by others to be a mammary cell-specific enhancer.
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PMID:Prolactin acts on the extreme 5' portion of MMTV LTR involving a mammary cell-specific enhancer. 827 23

To investigate the possible direct/indirect role of Human immunodeficiency virus (HIV) as a cofactor in human papillomavirus (HPV) oncogenesis, cotransfection experiments were carried out in which a recombinant plasmid containing the HPV16 long control region (LCR) linked to the chloramphenicol acetyltransferase (CAT) gene was cotransfected into cultured cells with a plasmid expressing HIV-1 Tat protein. Tat expression efficiency and transactivation activity were evaluated in different cell lines by cotransfecting plasmids containing the HIV tat gene and HIV LTR-driven CAT-coding sequences. HeLa and CaSki cell lines represented the most appropriate recipient cells for Tat-directed transactivation of both the HIV LTR and the HPV LCR promoters. Furthermore, HIV tat was transfected into HeLa cells (containing 10-20 copies per cell of HPV18), and HPV18 E7 protein expression was evaluated by a radioimmunoprecipitation assay using polyclonal antibodies against the E7 protein. Our results show that the Tat protein can transactivate the HPV LCR and increase HPV18 E7 expression in HeLa cells.
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PMID:Human immunodeficiency virus type 1 tat gene enhances human papillomavirus early gene expression. 829 82

Molecular interactions between the herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV) were investigated in U937 cells. Nonpermissive HSV-1 infection of U937 cells activated HIV as determined in transient expression assays with hybrid constructions containing the HIV-LTR-directed chloramphenicol acetyltransferase gene. Activation was independent of kappa B-enhancer elements whereas these elements were required for HSV-1-mediated activation in another cell line (C6). kappa B-binding proteins were induced in U937 cells by HSV-1 infection. Four species (45, 55, 75, and 75/80 kDa) were identified by DNA-protein cross-linking. Methylation interference analysis defined close contact only with the third residue of the previously established critical contact triplet GGG. Transient expression assays using mutants in HIV-LTR revealed the presence a cis-response element (GGTCA palindrome) in the negative regulatory region.
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PMID:NF-kappa B-binding proteins induced by HSV-1 infection of U937 cells are not involved in activation of human immunodeficiency virus. 838 Jun 62

The human immunodeficiency virus type 1 long terminal repeat, HIV-1-LTR, contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression. Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription. To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation, 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens. We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells. Additionally, Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase (CAT) gene. Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells. These studies suggest that, by inhibiting AP-1 activation, Nef may play a role in regulating HIV-1 gene expression in infected T-cells.
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PMID:HIV-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells. 848 Apr 25

CD8+ T lymphocytes of HIV-1-infected individuals can efficiently suppress HIV-1 replication in CD4+ T lymphocytes. To elucidate the molecular events underlying this suppression, we have used the HIV-1 LTR directing the chloramphenicol acetyltransferase gene (CAT) in transient transfection assays using human Jurkat T cells. In addition to supernatants of patient CD8+ T lymphocytes (CD4+ > 350/microliters), supernatant of a T cell clone derived by Herpesvirus saimiri (HVS)-mediated transformation of CD8+ T lymphocytes of a patient demonstrating inhibition of virus replication were examined. Similar levels of inhibition of LTR-mediated gene expression in response to Tat or mitogenic activation with phorbol ester and calcium ionophore were observed by supernatants of both sources. The inhibitory effect of CD8+ T lymphocytes was not exclusive to lentiviral LTRs since transcription of both the HTLV-I LTR and RSV LTR in response to mitogen was effectively inhibited. In examination of the influence of CD8+ T cell-derived supernatant on NF kappa B-mediated activation, a dimer of the HIV-1 NF kappa B elements directing CAT was markedly inhibited by supernatants of both patient CD8+ lymphocytes and the HVS-derived CD8+ clone. Thus the inhibitory nature of CD8+ T lymphocytes appears not to be specific to lentiviral promoters and may mediate an inhibitory effect via the NF kappa B element.
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PMID:Suppression of activation of the human immunodeficiency virus long terminal repeat by CD8+ T cells is not lentivirus specific. 857 88

A 25-residue peptide representing the membrane targeting N-terminal splice region of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1) was synthesized, and its structure was determined by 1H NMR. Two independently folding helical regions were identified, separated by a highly mobile "hinge" region. The first helical region was formed by an N-terminal amphipathic alpha-helix, and the second consisted of multiple overlapping turns and contained a distinct compact, hydrophobic, tryptophan-rich domain (residues 14-20). Chimeric molecules, formed between the N-terminal region of RD1 and the soluble bacterial protein chloramphenicol acetyltransferase, were used in an in vitro system to determine the features within the splice region that were required for membrane association. The ability of RD1-chloramphenicol acetyltransferase chimera to become membrane-associated was not affected by deletion of any of the following regions: the apolar section (residues 2-7) of the first helical region, the polar part of this region together with the hinge region (residues 8-13), or the polar end of the C-terminal helical region (residues 21-25). In marked contrast, deletion of the compact, hydrophobic tryptophan-rich domain (residues 14-20) found in the second helical region obliterated membrane association. Replacement of this domain with a hydrophobic cassette of seven alanine residues also abolished membrane association, indicating that membrane-association occurred by virtue of specific hydrophobic interactions with residues within the compact, tryptophan-rich domain. The structure of this domain is well defined in the peptide, and although the region is helical, both the backbone and the distribution of side chains are somewhat distorted as compared with an ideal alpha-helix. Hydrophobic interactions, such as the "stacked" rings of residues Pro14 and Trp15, stabilize this domain with the side chain of residue Leu16 adopting a central position, interacting with the side chains of all three tryptophan residues 15, 19, and 20. These bulky side chains thus form a hydrophobic cluster. In contrast, the side chain of residue Val17 is relatively exposed, pointing out from the opposite "face" of the peptide. Although it appears that this compact, tryptophan-rich domain is responsible for membrane association, at present the target site and hence the specific interactions involved in membrane targeting by the RD1 splice region remain unidentified.
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PMID:Determination of the structure of the N-terminal splice region of the cyclic AMP-specific phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and identification of the membrane association domain using chimeric constructs. 866 81

HIV-1 infection has been documented in rabbits, but infection proceeds slowly in this species. Human and rabbit cell lines were compared in order to identify barriers to efficient HIV-1 infection of rabbit cells. A direct comparison of human and rabbit CD4 as receptor for HIV-1 indicated that the rabbit CD4 homolog did not function well even when expressed by human cells. Examination of viral RNA production indicated that the major HIV transcripts were produced in HIV-infected rabbit cells, but were present at levels significantly lower than those found for human cells. Ability of HIV-1 LTRs to direct protein expression in human and rabbit cells was compared using gene constructs with the chloramphenicol acetyltransferase (cat) gene flanked by HIV-1 LTRs. Chloramphenicol acetyltransferase protein expression was equivalent in rabbit and human cell lines transfected with the HIV-1/CAT constructs and cotransfections with the HIV-1 tat gene led to similar increases in CAT expression. Subsequent transfections with an infectious molecular HIV clone yielded approximately equal levels of HIV protein expression in rabbit and human cell lines, suggesting that major barriers to virus production in rabbit lines exist at steps prior to transcription of the viral genome. Because HTLV-I replicates with high efficiency in rabbit cells, a chimeric virus clone was constructed consisting of the 5' portion of HIV-1 through the nef coding sequence followed by the 3' HTLV-I LTR. Transfection of most rabbit cell lines with the chimera produced levels of p24gag protein higher than those transfected with the parent HIV-1 clone. By contrast, the unmodified HIV clone replicated more efficiently in all human cell lines tested.
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PMID:Replication of HIV type 1 in rabbit cell lines is not limited by deficiencies in tat, rev, or long terminal repeat function. 867 93

Indoleamine 2,3-dioxygenase (INDO) is the rate-limiting enzyme in the catabolism of the essential amino acid L-tryptophan. It is induced strongly in many cell lines following interferon-gamma treatment. We report the cloning and characterization of the full-length human INDO promoter. This promoter is 1,245 base pairs long and includes two interferon-stimulated response elements (ISRE) separated by an approximately 1-kilobase sequence. The presence of these two ISREs is critical for maximum INDO promoter activity (50-fold induction). When the ISREs are present in two separate fragments cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter vector, the INDO promoter activity drops significantly (7-fold induction). 5' end deletions of the wild type promoter sequence indicate that removal of the ISRE (ISRE1) at position -1126 reduces the induction level to approximately 25-fold. This activity does not change appreciably when the promoter is deleted down to position -241. Furthermore, site-directed mutagenesis of ISRE1 also decreases the promoter activity in a similar way. When ISRE1 is kept intact, deletion of the second ISRE (ISRE2) at position -111 leads to only 11-fold induction of the promoter. A similar result is obtained when substitution mutations are introduced in ISRE2. Deletion of a 748-base pair sequence between the two ISREs only shows a slight decrease in the INDO promoter activity. These data indicate that the two ISRE sequences are required for the full transcriptional induction of the interferon-gamma-inducible human INDO gene. INDO activity is not induced in the hepatic cell line HepG2. An analysis of INDO-CAT activity in this cell line indicated that the lack of INDO activity was at the transcriptional level and could reflect either the presence of a repressor or lack of a transcription factor. This lack of induction could be correlated with a truncated or unstable IRF-1. However, the levels of IRF-2, JAK2, and STAT 91 were similar in both ME180 and HepG2 cells.
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PMID:Importance of the two interferon-stimulated response element (ISRE) sequences in the regulation of the human indoleamine 2,3-dioxygenase gene. 870 90

Human lymphoid cell lines were transfected with HIV-1-LTR-CAT DNA and permanently transformed cell lines were obtained. These transformed cell lines were treated with 0.01 mM H2O2 for 25 days and the chloramphenicol acetyltransferase (CAT) activities of these cell lines were measured. The CAT activities of transformed cell lines were latent in normal culture conditions, but were activated by retreatment with 0.2 mM H2O2 for 1 h. On treatment with 0.05 mM H2O2 for 1 h, the CAT activity of these cell lines maintained in normal conditions remained latent, whereas cell lines pretreated with 0.01 mM H2O2 for 25 days were greatly activated by this treatment. Here, the HIV-1 promoter seemed latent in normal culture conditions, but it could be activated by a comparatively low concentration (0.05 mM) of H2O2 after treatment with a dilute H2O2 (0.01 mM) for about 1 month. Many patients infected with human immunodeficiency virus 1 (HIV-1) show a long latent period before development of AIDS. During this latent period, their infected cells may be subjected to oxidative stress due to metabolism and physical movement. The present results indicate that oxidative stress may cause activation of the HIV-1 promoter in patients with latent HIV-1.
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PMID:Sensitization of the HIV-1-LTR upon long term low dose oxidative stress. 870 77


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