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 constructed stable DNA-transfected LTK+ cell lines containing two different coselected hybrid interferon (IFN) genes driven by the usually strong and constitutive promoter from the immediate-early 94K protein (IE94) gene of simian cytomegalovirus. Surprisingly, and unlike hybrid IE94-chloramphenicol acetyltransferase gene constructs, both of the IE94-IFN genes (one with and one without the complex spliced intron region) produced relatively low basal titers of biologically active human IFN in the mouse cell lines. However, IFN expression could be stimulated up to 120-fold by superinfection with herpes simplex virus (HSV), although not with cytomegalovirus. To examine the mechanism of this unexpected HSV induction process, we measured the levels of both IE94-IFN mRNA and IFN protein produced under various infection protocols. Compared with similar previously characterized cell lines containing hybrid IFN genes under the control of HSV IE or delayed-early (DE) promoters, activation of IFN expression first occurred at an intermediate time. Both IE94-IFN cell lines also produced an unusual pattern of response to infection with the HSV IE regulation-deficient mutants tsK and tsB7: stimulation of IFN synthesis occurred in the absence of a functional HSV IE175 (or ICP4) gene product, but did not occur in the absence of uncoating of virus capsids. Cycloheximide treatment (without virus infection) also gave a rapid 30-fold increase in steady-state levels of correctly initiated mRNA from both types of IE94-IFN hybrid genes, but had no effect on cells containing the IE175-IFN construct. Therefore, we conclude that the use of the IE94-IFN constructs identifies a novel HSV regulatory response that requires a previously unrecognized function of HSV and does not involve either IE175 or the pre-IE "virion factor" trans-activators that are known to stimulate transcription of HSV IE and DE genes, respectively.
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PMID:Novel induction by herpes simplex virus of hybrid interferon gene transcripts driven by the strong cytomegalovirus IE94 promoter. 243 69

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, we examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. Analysis of promoter deletion mutants indicated that the 5' minimal sequence required for activation is -61 from the CAP site (+1) and that an 8-base-pair sequence located at -51 to -58 is necessary for activation of the pp65 promoter. This sequence is repeated once at +93 and is found as an inverted repeat at +67. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.
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PMID:Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products. 253 31

Prior studies have demonstrated that a small proportion of blood lymphocytes from patients with human cytomegalovirus (HCMV) infection express only the viral immediate-early (IE) genes (L. Einhorn and A. Ost, J. Infect. Dis. 149:207-214, 1984; G. P. A. Rice, R. D. Schrier, and M. B. A. Oldstone, Proc. Natl. Acad. Sci. USA 81:6134-6138, 1984). The present studies demonstrate that the IE genes of HCMV are transcribed in Jurkat cells (T lymphocytes) only after activation of the cells with mitogens. Transcription of the IE genes is from an upstream enhancer promoter-regulatory region containing several different repeated sequence motifs. Chimeric plasmids were constructed with just a single copy or three copies of a synthetic oligonucleotide sequence of either the 16-, 18-, 19-, or 21-base-pair (bp) repeat elements upstream of the minimal wild-type promoter sequence to drive expression of the indicator gene, chloramphenicol acetyltransferase (CAT). The 18- or 19-bp motifs in the enhancer region were found to be important in mediating the effect of the mitogens. However, the CAT activity detected with the 19-bp repeat was always significantly higher than that found with the 18-bp repeat. There was an additive effect by multiple copies of the 18- or 19-bp repeat sequences on gene expression. The 19-bp repeat contains a sequence identical to that described for a cyclic AMP (cAMP) response element, and plasmids containing only this sequence and the minimal promoter sequences upstream of the CAT gene respond to agents which increase intracellular cAMP. Functional cAMP response elements are present in the wild-type promoter-regulatory region and are associated with the 19-bp repeat sequences. It is proposed that activation of lymphocytes results in expression of the IE genes of HCMV, in part via the activation of cellular trans-acting factors which interact with the 18- and 19-bp motifs in the HCMV IE promoter-regulatory region. The 19-bp repeat is the major contributor to the strength of this enhancer-containing promoter-regulatory region.
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PMID:The promoter-regulatory region of the major immediate-early gene of human cytomegalovirus responds to T-lymphocyte stimulation and contains functional cyclic AMP-response elements. 254 10

We have studied the regulation of expression of a major human cytomegalovirus (HCMV) early transcription unit located within the long repeat of the strain AD169 genome. This region specified a 2.7-kilobase RNA which underwent its largest increase in abundance between 8 and 14 h postinfection. To study the regulation of this gene, its promoter was cloned 5' of the gene for chloramphenicol acetyltransferase (CAT) for use in transient expression assays. A construct containing 651 base pairs of upstream sequence and 54 base pairs of leader sequence was transfected into human fibroblast cells, followed by HCMV infection. Analysis of the steady-state levels of RNA expressed from this hybrid gene indicated that it accumulated with the same kinetics as the authentic viral transcript early in the infection. Cotransfection of human fibroblasts with the 2.7-kilobase RNA promoter-CAT construct and plasmids containing different HCMV immediate-early (IE) genes showed that the region of the HCMV genome encoding the transcription units corresponding to IE1 and 2 and the 5' end of IE3 is capable of stimulating promoter activity but not to the same extent as HCMV infection. To define important cis-acting regulatory elements in the promoter, a series of 5' deletion mutants was constructed. Transient expression analysis showed a stepwise reduction in inducible CAT activity, suggesting the presence of multiple regulatory sites. To further characterize the nature of these sites, we used gel mobility shift assays to study DNA-protein interactions occurring within this promoter sequence. With nuclear extracts prepared from HeLa cells as well as from infected and uninfected human foreskin fibroblasts, we found specific binding of a cellular factor to a region of the promoter important in HCMV inducible activity. This region contains a palindromic octamer with homology to the binding site of the cellular factor USF/MLTF.
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PMID:Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene. 255 58

In preparation for studies using gene transfer, we have identified transcriptional control elements which are active in primary rat hepatocytes. We used plasmids which were constructed so that the promoter or enhancer of interest initiated transcription of the bacterial chloramphenicol acetyltransferase (CAT) gene. Plasmids were introduced into primary rat hepatocytes in culture, into Hep G2 cells and other human and animal cell lines and into bone marrow stromal cells, and CAT activity was assayed after 48 hr. In primary rat hepatocytes, the highest CAT activity was obtained with plasmids carrying the Rous sarcoma virus long terminal repeat (pRSVCAT), or the SV40 early region promoter and enhancer (pSV2CAT). Hepatocytes carrying the murine cytomegalovirus immediate early promoter (pUCRNmCMVX/HCAT) also had appreciable CAT activity. No CAT activity was detected in rat hepatocytes carrying pSVOCAT (a promoterless construct), pUCRNtKCAT (herpes simplex thymidine kinase gene promoter), pLPVCAT (lymphocytotrophic papovavirus promoter) and pHBV1CAT (hepatitis B virus enhancer and core gene promoter). Therefore, for future studies of gene transfer in primary rat hepatocytes, the Rous sarcoma virus long terminal repeat or the SV40 early region promoter and enhancer can be effectively used to drive gene expression. Hep G2 cells carrying pHBV1CAT had high CAT activity. Hep G2 cells carrying pHBV2CAT (similar to pHBV1CAT, but with the hepatitis B virus sequences in reverse orientation with respect to the CAT sequences) and pHBV3CAT (similar to pHBV2CAT, but hepatitis B virus sequences are separated from the CAT sequences by about 700 bases) also expressed CAT activity, but not as strongly as with pHBV1CAT.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue-specific activity of heterologous viral promoters in primary rat hepatocytes and Hep G2 cells. 280 56

Herpes simplex virus 1 (HSV-1) infection induces transcription of the chloramphenicol acetyltransferase (CAT) gene directed by the long terminal repeat (LTR) of human immunodeficiency virus (HIV) in both transiently and permanently transfected cells containing the HIV-LTR/CAT hybrid gene. To define the mechanism by which HSV-1 stimulates the HIV LTR, we examined the effects of isolated regulatory genes from HSV-1. The results of cotransfection assays with the immediate-early (IE) genes of HSV-1, IE110 (ICP0) and IE175 (ICP4), showed that the IE110 protein, either alone or in combination with the IE175 protein, can activate the HIV LTR. Cotransfection with the IE175 gene alone or with the Vmw65 gene (coding for a virion transcription factor) alone did not lead to HIV-LTR activation. The lack of requirement for the IE175 or Vmw65 gene products in transient-expression assays was confirmed in permanent cell lines containing the HIV-LTR/CAT hybrid gene by using temperature-sensitive mutants defective in the IE175 gene product or in uncoating functions. By deletion analysis, we localized a 73-bp-long region (positions -104 to -32) from the HIV LTR that responded to HSV-1 activation; when this region, which is distinct from the previously identified trans-activating responsive (TAR) region, was ligated to a heterologous, HSV-1-nonresponsive gene (alpha 4-interferon/CAT), it conferred inducibility by both HSV-1 infection and IE110/175 cotransfection. Both simian and human cytomegalovirus also induced the HIV-LTR/CAT hybrid gene. However, we failed to detect specific upstream sequence requirements for induction by cytomegalovirus. Our results indicate that infection with unrelated viruses can alter the expression of HIV in an infected cell.
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PMID:Activation of human immunodeficiency virus by herpesvirus infection: identification of a region within the long terminal repeat that responds to a trans-acting factor encoded by herpes simplex virus 1. 282 60

Almost all homosexual patients with acquired immunodeficiency syndrome are also actively infected with human cytomegalovirus (HCMV). We have hypothesized that an interaction between HCMV and human immunodeficiency virus (HIV), the agent that causes acquired immunodeficiency syndrome, may exist at a molecular level and contribute to the manifestations of HIV infection. In this report, we demonstrate that the immediate-early gene region of HCMV, in particular immediate-early region 2, trans-activates the expression of the bacterial gene chloramphenicol acetyltransferase that is fused to the HIV long terminal repeat and carried by plasmid pHIV-CAT. The HCMV immediate-early trans-activator increases the level of mRNA from the plasmid pHIV-CAT. The sequences of HIV that are responsive to trans-activation by the HCMV immediate-early region are distinct from HIV sequences that required for response to the HIV tat. The stimulation of HIV gene expression by HCMV gene functions could enhance the consequences of HIV infection in persons with previous or concurrent HCMV infection.
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PMID:Immediate-early gene region of human cytomegalovirus trans-activates the promoter of human immunodeficiency virus. 282 1

The participation of human cytomegalovirus (HCMV) immediate early genes in the activation of the expression of adenovirus genes in trans (trans-activation) was examined. The initial strategy used was to determine the ability of HCMV genes to complement mutants of adenovirus E1a, an immediate early gene which encodes a trans-activator. The HCMV immediate early gene regions IE1 and IE2 complemented E1a-deficient mutants in three separate assays. IE1 and IE2 substituted for E1a in the synthesis of infectious adenovirus, late adenovirus RNA, and adenovirus DNA. Complementation by the IE2 gene region alone, but not by IE1 alone, was observed using the most discriminating assay, that for late adenovirus RNA synthesis. A role for both HCMV gene regions in positive transcriptional control was indicated by their ability to increase expression of chloramphenicol acetyltransferase (CAT) mediated by the adenovirus E2a promoter. The IE2 region alone activated CAT synthesis but IE1 alone had no detectable activity. Moreover, the activity of both gene regions was about 10-fold higher than that of IE2 alone. These data indicate that efficient complementation of E1a-deficient mutants and trans-activation of adenovirus early promoters involved the participation of both HCMV immediate early gene regions.
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PMID:Participation of two human cytomegalovirus immediate early gene regions in transcriptional activation of adenovirus promoters. 282 3

To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells. However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three- to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negative modulates expression in nonpermissive cells but positively influences expression in permissive cells.
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PMID:Negative and positive regulation by a short segment in the 5'-flanking region of the human cytomegalovirus major immediate-early gene. 282 27

The construction of a mammalian cell expression vector using human cytomegalovirus immediate early gene enhancer to initiate transcription of inserted coding sequences is described. The vector also carries Epstein-Barr virus EBNA-1 nuclear antigen gene, ori-P sequences and hygromycin B resistance gene hph from E. coli. The expression capacity of this construct was tested by inserting the chloramphenicol acetyltransferase (CAT) gene into the vector. The EBV-CAT construct was transfected into various cell lines and high levels of CAT activity were obtained in human and monkey cells. In these cells, the vector DNA also replicates as an extrachromosomal element having 1 to 20 copies per cell. In most cases, the vector copy number and the expression level of inserted gene was in positive correlation in different cell clones.
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PMID:An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences. 282 66


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