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)

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.
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PMID:Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene. 283 Apr 97

We examined the interaction of human immunodeficiency virus (HIV) and herpes group viruses. For this purpose, a chimeric plasmid (pLTR-CAT) was constructed in which the long terminal repeat (LTR) sequences derived from a molecular clone of HIV were fused to a bacterial chloramphenicol acetyltransferase gene (CAT). Transient expression assays in transfected tissue culture cells were used to monitor the activity of the LTR. Basal levels of CAT activity were measured in HeLa and human lung fibroblast (HLF) cells transfected with pLTR-CAT. When HeLa or HLF cells transfected with pLTR-CAT were infected with herpesviruses, HIV LTR-directed expression of the CAT gene was detected. An enhancement of the HIV LTR-directed expression of CAT was observed for herpes simplex virus (HSV)-1 and HSV-2, cytomegalovirus and varicella zoster virus. Enhanced CAT expression directed by the LTR was also shown by cotransfection of recombinant plasmids containing two non-overlapping regions of HSV-1, a fragment from HSV-2 which is non-colinear with the regions used from HSV-1, the immediate early gene of pseudorabies virus and the adenovirus early gene EIA. HIV LTR-directed expression may be a useful model for studying the effects on HIV of various infectious agents known to be present in individuals with AIDS or HIV infection.
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PMID:Transactivation of human immunodeficiency virus by herpesviruses. 283 May 74

The major immediate-early (IE) gene region mapping at coordinates 0.71 to 0.74 in the genome of human cytomegalovirus (HCMV) gives rise to a series of overlapping spliced IE mRNAs that are all under the transcriptional control of the complex IE68 promoter-enhancer region. We show here that one of the phosphorylated nuclear proteins encoded by this region behaves as a powerful but nonspecific trans-activator of gene expression. In transient chloramphenicol acetyltransferase (CAT) assay experiments with Vero cells all relatively weak heterologous target promoters tested, including those of herpes simplex virus IE175 and delayed-early genes, adenovirus E3, the enhancerless simian virus 40 early gene, and the human beta interferon gene, were stimulated between 30- and 800-fold by cotransfection with the HindIII C fragment of HCMV (Towne) DNA. In contrast, expression of the homologous HCMV IE68-CAT gene but not SV2-CAT was specifically repressed. Inactivation mapping studies of the effector DNA, together with dose-response comparisons with subclones from the region, revealed that an intact 7.1-kilobase sequence encompassing both the IE1 and IE2 coding regions (exons 1 to 5) in the major IE transcription complex was required for both the nonspecific trans-activation and autoregulatory responses. The IE1 coding region alone (exons 1 to 4) was inactive, but both functions were restored by insertion of the IE2 coding region (exon 5) in the correct orientation downstream from the IE1 coding region. Internal deletions or inserted terminator codons in IE1 (exon 4) still gave efficient trans-activation and autoregulation, whereas the insertion of terminator codons in IE2 (exon 5) abolished both activities. Finally, IE2 (exon 5) sequences only (under the direct transcriptional control of the strong simian CMV IE94 promoter) were still able to specifically down regulate IE68-CAT expression but failed to exhibit trans-activation properties. Therefore, the IE2 gene product(s) of HCMV appear likely to be key control proteins involved in gene regulation during HCMV infection.
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PMID:trans-activation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus. 283 79

Herpes simplex virus type 1 (HSV-1) encodes several alpha (immediate-early) gene products that modulate gene expression during viral replication. We report here that the alpha protein ICP27 specifically stimulates expression of a later viral gene, that encoding glycoprotein B (gB). Using temperature-sensitive viral mutants, the effect of ICP27 on HSV-1 protein synthesis was examined at early times after infection or at later times when viral DNA replication was inhibited. Under these conditions, the expression of gB showed a marked dependence on the presence of functional ICP27, whereas several other beta and gamma 1 genes showed a lesser dependence. It was also noted that cells infected with ICP27 temperature sensitive mutants at the nonpermissive temperature showed a reduction in the electrophoretic mobility of the alpha protein ICP4. To examine the mechanism by which ICP27 stimulated gB expression, a plasmid was constructed in which the promoter-regulatory region of the gB gene was fused to the gene encoding chloramphenicol acetyltransferase (CAT). CAT expression from this plasmid was induced significantly by ICP27 expressed from a cotransfected plasmid. Induction of CAT activity by ICP27 correlated well with an increase in the amount of CAT transcripts initiated from the transcriptional start site of the gB gene. The transactivating activity of ICP27 was specific for the gB promoter-regulatory region, as expression from several other HSV-1 promoter-CAT chimeric genes was not stimulated by ICP27. The DNA sequences which conferred the response to ICP27 mapped within 175 base pairs upstream and 41 base pairs downstream of the gB transcriptional start site. Our results suggest that the full expression of gB and perhaps other viral genes during HSV-1 infection requires the combined action of multiple viral transactivators.
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PMID:Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27. 284 77

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down
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PMID:The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. 284 67

On the basis of experiments with mutant virus and transfection with isolated genes, the herpes simplex virus immediate-early gene product ICP4 is known to positively regulate the transcription of viral early and late genes and negatively regulate expression from its own promoter. Binding of ICP4 to DNA sequences in several viral genes has been reported, yet the significance of ICP4-DNA interaction in transcriptional activation remains unclear. We have studied this problem by using the early glycoprotein D (gD) gene, which possesses a binding site at approximately -100 relative to the RNA initiation site. We linked this promoter and various mutant constructs to the chloramphenicol acetyltransferase gene in order to measure promoter activity in transient transfections both in the presence and in the absence of an ICP4-encoding plasmid. The natural promoter was activated 3.3-fold, and a deletion construct lacking the binding site was activated minimally (1.7-fold). Constructs containing multiple tandem repeats of the binding site (three or five inserts) demonstrated higher expression in the presence of ICP4 than did the natural promoter while retaining low levels of expression when unstimulated. Gel mobility shift assays and DNase I footprinting analyses indicated that ICP4 associated with multiple binding sites. In vitro transcription from a gD promoter construct containing multiple binding sites showed increased RNA synthesis in the presence of partially purified ICP4. These data provide the first direct evidence that binding of ICP4 to a specific DNA sequence in the gD gene contributes to activation of transcription.
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PMID:Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene. 284 78

We have constructed a transient expression vector (pCAL) containing two reporter genes, chloramphenicol acetyltransferase (CAT) and beta-galactosidase (beta-gal) for use in studying herpes simplex virus type 1 (HSV-1) promoter activity in mammalian cells. The construct was designed to be useful in analyzing the simultaneous expression from two different promoters. To test the utility of the vector, we used three HSV-1 promoters that had been characterized previously by workers in this laboratory. Two are early (beta) promoters, for alkaline exonuclease and deoxyuridine triphosphate nucleotidohydrolase; the third promoter controls the major capsid protein transcript and is late (beta gamma). The two different kinetic classes of promoters were ligated in a divergent orientation into pCAL and transfected into rabbit skin fibroblast. Transfected cells were then superinfected with low multiplicities of HSV-1; 18 hr later, we observed the simultaneous expression of both marker genes under control of the respective promoters. The usefulness of such a transient expression reporter vector is discussed.
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PMID:A bi-functional reporter plasmid for the simultaneous transient expression assay of two herpes simplex virus promoters. 285 22

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid.
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PMID:Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor. 289 22

Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or by indirect effects of steroid on the hypothalamus. To study direct estrogen regulation of the rat luteinizing hormone beta (LH beta) gene, we performed estrogen receptor-DNA binding studies and transient expression gene transfer experiments. Nitrocellulose filter binding studies were performed with purified estrogen receptor from calf uterus and labeled fragments of the LH beta gene. Dose-dependent specific binding to receptor occurred only with LH beta gene fragments containing a common 284-base region from -1388 to -1105 bases upstream from the transcriptional start site. This DNA region contained a 15-base imperfect palindromic region (GGACACCATCTGTCC) with sequence similarity to other estrogen-responsive elements. Biological function was tested by inserting portions of the 5'-flanking region of the gene next to the herpes simplex virus thymidine kinase promoter fused to the chloramphenicol acetyltransferase gene (LH beta-tkCAT) and performing gene transfer experiments with the pituitary GH3 cell line. Promoter activity in LH beta-tkCAT constructs containing LH beta gene sequences from bases -2013 to -613, or from bases -1388 to -613 in either orientation, exhibited stimulation with 17 beta-estradiol (E2) treatment; in contrast, constructs containing bases -885 to -613 were not regulated by E2. Positive regulation by E2 exhibited dose- and time-dependent stimulation, with a maximum 2- to 6-fold effect achieved after 48 h of treatment with 10(-8) M E2. The estrogen receptor appeared to be required for this biological response. Stimulation of LH beta-tkCAT constructs did not occur in L cells with undetectable levels of E2 receptor, but did occur after cotransfection of an LH beta-tkCAT construct and an expression vector containing the human estrogen receptor cDNA. These studies demonstrate that a 5'-flanking region of the rat LH beta gene can bind to the estrogen receptor and that this region can confer hormonal responsiveness to a heterologous promoter. Thus, positive steroid regulation of luteinizing hormone may occur directly on the pituitary at the level of the LH beta gene.
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PMID:An upstream region of the rat luteinizing hormone beta gene binds estrogen receptor and confers estrogen responsiveness. 290 46

We reconstructed the regulated induction of delayed-early (DE) transcription that occurs during herpes simplex virus (HSV) infection by using a transient expression system in which recombinant target genes were cotransfected into Vero cells together with intact activating genes. Plasmids containing cloned HSV-1 or HSV-2 immediate-early (IE) genes stimulated by up to 100-fold the expression from recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the DE promoter/regulatory region from the genes for an HSV-2 38,000-molecular-weight (38K) protein and the HSV-1 thymidine kinase. This activation was specific to hybrid genes containing DE regulatory regions since no significant increases in expression were observed in cotransfection experiments with the CAT gene without any promoter region or under the control of a number of other regulatory regions, including an HSV-1 IE regulatory region, the complete or enhancerless early regulatory region of simian virus 40, and an inducible cellular promoter/regulatory region. By using a variety of cotransfected plasmids containing individual or different combinations of HSV-1 or HSV-2 IE genes, we show that of the five known IE genes, two, those coding for the 175K and 110K polypeptides, each possessed the ability to stimulate expression from both DE promoters. Cleavage of the input plasmids within the known coding regions for the 175K and 110K proteins abolished stimulation of DE/CAT gene expression, whereas cleavage outside the coding regions had no effect on stimulation. We conclude that stimulation of CAT expression occurred exclusively by a transactivation mechanism in which the products encoded by these IE genes acted on the DE hybrid constructs at the transcription level. No transcriptional stimulatory function was demonstrated for the IE 68K and 63K proteins, although our results indicate that the IE 12K protein may augment the DE stimulatory activity of the 175K and 110K proteins.
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PMID:Evidence for a direct role for both the 175,000- and 110,000-molecular-weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters. 298 86


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