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 phosphoprotein synapsin I is expressed exclusively in neuronal cells. We are interested in elucidating the promoter sequences involved in cell type-specific expression of the synapsin I gene. The PC12 cell line expresses the 3.4 kb and 4.5 kb synapsin I mRNAs and is used to analyze cell type-specific gene expression. A series of deletion fragments of the rat synapsin I gene promoter were fused to the promoterless reporter gene encoding bacterial chloramphenicol acetyltransferase (CAT) for transfection analysis in PC12 cells and in HeLa cells, which do not express the gene. A -349 bp to +110 bp rat synapsin I promoter fragment contains a positive regulator, shown to be 33-times more active in PC12 cells than HeLa cells. Transfection of reporter plasmids containing up to 4.4 kb of rat synapsin I gene promoter sequences exhibit significantly reduced CAT activity in PC12 cells. The reduction in CAT expression was attributed to a negative regulator located between -349 bp and -1341 bp in the rat synapsin I promoter. Our results suggest that both positive and negative-acting sequence elements regulate cell type-specific expression of the rat synapsin I gene.
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PMID:Positive- and negative-acting promoter sequences regulate cell type-specific expression of the rat synapsin I gene. 166 26

The 5'-terminal region of the rat gene for the neuron-specific phosphoprotein, synapsin I, was isolated and sequenced. It comprises 1472 nucleotides (nt) of 5'-flanking sequence, 507 nt of the first exon, and 242 nt of the first intron. A single transcription start site was mapped by primer extension and S1 nuclease analysis. A sequence of 340 nt upstream from the transcription start site and the first exon are G+C-rich and enriched in CpG dinucleotides, resembling a CpG island. The 5'-flanking sequence lacks TATA and CAAT consensus elements but contains a consensus motif for the cAMP-responsive element. Furthermore, we notice two potential consensus motifs which are also found in corresponding positions in the genes for the nerve growth factor receptor and the 68-kDa neurofilament protein. The 5'-terminal region of the human synapsin I gene was also cloned and sequenced. A high degree of sequence conservation between rat and human is found in the upstream 340 nt that coincides precisely with the G+C-rich domain and includes the consensus elements, and throughout the first exon including the untranslated sequence. Sequence conservation is also observed further upstream and at the beginning of the first intron. In a transient chloramphenicol acetyltransferase expression assay, 5'-flanking sequences of the rat synapsin I gene function as strong promoters in neuroblastoma cells, but not in fibroblastoid cells. 225 nt of 5'-flanking sequence and 105 nt of 5'-untranslated sequence are sufficient for cell-type specific transcription in this assay.
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PMID:The 5'-flanking region of the synapsin I gene. A G+C-rich, TATA- and CAAT-less, phylogenetically conserved sequence with cell type-specific promoter function. 211 19

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

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection.
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PMID:The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription. 300 59

At early times after infection with herpes simplex virus, transcription from beta-promoters is initiated only in the presence of a functional 174,000 Mr phosphoprotein (ICP4), encoded by an immediate early (alpha) gene (IE4). A transient expression assay was used to analyze the requirement for two (ICP4 and ICP0) of the five alpha-gene products in the transcriptional regulation of model alpha and beta-gene promoters. These studies reveal that cells cotransfected with plasmids containing the alpha-gene sequences for infected cell proteins (ICPs) 4 and 0 and a thymidine kinase (TK, a beta-gene) gene or the thymidine kinase promoter fused to a chloramphenicol acetyltransferase (CAT) cassette accumulate 10 to 20-fold more RNA or exhibit 10 to 20-fold more CAT activity than cells cotransfected with a plasmid encoding either alpha-gene protein and a thymidine kinase indicator gene. Functional ICP4 is required for enhanced transcriptional activation in the transient expression assay system. It is also required for the uniform dispersal of ICP0 throughout the nucleus as shown by immunofluorescence staining analysis of transfected cells. Two alpha-promoter-CAT fusions were used as targets to study what effects ICP4, ICP0 and Vmw65 (the virion-associated alpha-gene transactivator) have on expression from alpha-promoters that contain all of the sequences that confer alpha-gene regulation, or only the core sequence governing basal level expression. We conclude that ICP4 can activate alpha-gene expression from the core sequence and, depending on its abundance, activate or repress expression from a promoter containing the sequences required for alpha-gene regulation. Independent of these alpha-regulatory sequences cotransfection with low levels of sequences encoding both ICP0 and ICP4 activate expression. At higher ratios of effector (both ICP4 and ICP0) the target accumulation of CAT activity decreases. Although a ts allele of IE4 (cloned from the mutant virus tsK) does not activate alpha-gene expression it can enhance the ability of ICP0 to activate a target containing alpha-regulatory sequences. Virus studies involving tsK support the conclusion that functional ICP4 is required to activate beta-promoters and to repress expression from alpha-promoters and help to explain the pleiotropic effects of the tsK mutation. These analyses have also revealed the presence of a novel RNA species that overlaps the sequences encoding ICP0. Our results suggest that co-ordinate regulation of HSV gene expression is mediated by the functional interaction of at least two alpha-gene products, ICP0 and ICP4.
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PMID:Co-ordinate regulation of herpes simplex virus gene expression is mediated by the functional interaction of two immediate early gene products. 302 83

Anti-c-Src and anti-phosphotyrosine immunoprecipitates from receptor-like protein tyrosine phosphatase alpha (PTP alpha)-transfected and control rat embryo fibroblasts contain a 39-kDa phosphoprotein (p39) whose phosphorylation is enhanced by PTP alpha expression. The p39 that co-immunoprecipitates with c-Src has been identified as c-Jun by immunological and functional criteria; it is recognized by several different anti-c-Jun antibodies and binds to a c-Jun recognition element-containing oligonucleotide. Whereas the association of c-Src and c-Jun is unexpected, it may be of significance in PTP alpha signaling since we have previously demonstrated that c-Src is activated by PTP alpha (Zheng, X. M., Wang, Y., and Pallen, C. J. (1992) Nature 359, 336-339. Examination of c-Jun activity in these fibroblasts demonstrates that c-Jun DNA binding activity and c-Jun-mediated transcription of a chloramphenicol acetyltransferase reporter gene are elevated in PTP alpha-expressing cells. In addition to c-Jun activation, mitogen-activated protein kinase is activated in PTP alpha-expressing cells and translocated to the nuclei of these cells. The nuclear localization of activated mitogen-activated protein kinase and c-Jun suggests that their activation represents downstream events in the receptor-like PTP alpha-initiated signaling pathway(s).
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PMID:Expression of receptor-like protein tyrosine phosphatase alpha in rat embryo fibroblasts activates mitogen-activated protein kinase and c-Jun. 752 77

Synapsin II is a neuron-specific phosphoprotein that selectively binds to small synaptic vesicles in the presynaptic nerve terminal. Here we report the cloning and sequencing of the 5'-flanking region of the human synapsin II gene. This sequence is very GC-rich and lacks a TATA or CAAT box. Two major transcriptional start sites were mapped. A hybrid gene consisting of the Escherichia coli chloramphenicol acetyltransferase gene under the control of 837 base pairs of the synapsin II 5'-upstream region was transfected into neuronal and nonneuronal cells. While reporter gene expression was low in neuroblastoma and non-neuronal cells, high chloramphenicol acetyltransferase activities were monitored in PC12 pheochromocytoma cells. However, there was no correlation between reporter gene expression in the transfected cells and endogenous synapsin II immunoreactivity. Using DNA-protein binding assays we showed that the transcription factors zif268/egr-1, polyoma enhancer activator 3 (PEA3), and AP2 specifically contact the synapsin II promoter DNA in vitro. Moreover, the zif268/egr-1 protein as well as PEA3 were shown to stimulate transcription of a reporter gene containing synapsin II promoter sequences. In the nervous system, zif268/egr-1 functions as a "third messenger" with a potential role in synaptic plasticity. PEA3 is expressed in the brain and its activity is regulated by proteins encoded from non-nuclear oncogenes. We postulate that zif268/egr-1 and PEA3 couple extracellular signals to long-term responses by regulating synapsin II gene expression.
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PMID:The human synapsin II gene promoter. Possible role for the transcription factor zif268/egr-1, polyoma enhancer activator 3, and AP2. 759 48

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 have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (beta-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from -609 to -41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from -40 to +106 are sufficient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.
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PMID:Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome. 808 94

Serum amyloid A (SAA) is a plasma protein whose synthesis is markedly increased in the liver during the inflammatory process. Previous analysis of SAA promoter function implicated the involvement of the CCAAT/enhancer-binding protein (C/EBP) in controlling this process. In this study, using antibodies against three C/EBP isoforms in DNA-binding and Western blot (immunoblot) assays, we found that in response to inflammatory signals, both C/EBP-delta and C/EBP-beta are induced and that their interactions with the SAA promoter element are necessary for the increased SAA gene expression. Cotransfections of liver cells with an SAA promoter-linked reporter chloramphenicol acetyltransferase gene and murine sarcoma virus-expressed C/EBP-delta or C/EBP-beta confirm such phenomena. The increased transactivating ability in the presence of the cellular phosphatase inhibitors okadaic acid and sodium orthovanadate, coupled with the observation that dephosphorylation severely inhibits the DNA-binding ability in vitro, implicates a role of phosphorylation in the regulation of the activities of the C/EBP-delta isoform. Consistent with these findings, we have detected higher levels of DNA-binding activity of C/EBP-delta prepared from cells treated with phosphatase inhibitors. We also present evidence that C/EBP-delta is a phosphoprotein. These results suggest that C/EBP-delta is regulated by phosphorylation and, in conjunction with C/EBP-beta, is one of the major proteins responsible for the increased transcription of the SAA gene in response to inflammatory stimuli.
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PMID:Serum amyloid A gene expression under acute-phase conditions involves participation of inducible C/EBP-beta and C/EBP-delta and their activation by phosphorylation. 819 68


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