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)

Transient expression of the chloramphenicol acetyltransferase (CAT) gene under the control of simian virus 40 (SV40)y Moloney murine leukemia virus, human T cell leukemia virus, and cytomegalovirus promoters was stimulated by the differentiation of F9 stem cells into primitive endoderm, but repressed again by further differentiation into visceral endoderm. Deletion mutants of the SV40 enhancer showed that a similar set of motifs is critical for CAT expression at all stages of F9 differentiation, but differentiation dependency was observed even in their absence. The stability of transient gene expression under the control of the SV40 promoter was markedly dependent on F9 differentiation. Appreciable expression was detected even in undifferentiated F9 cells immediately after gene transfection, was maximal at 12 h and declined rapidly thereafter. On the other hand, expression in primitive endoderm increased until 72 h. The decline was accelerated again in visceral endoderm. This shift was somewhat specific to the virus promoter since CAT expression in undifferentiated F9 cells under the control of the elongation factor 1 alpha promoter was more stable than for virus promoters tested. Thus, the change in stability of expression is important for differentiation-dependent virus promoter activity.
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PMID:Transient gene expression by SV40 promoter characterizes sequential differentiation of embryonal carcinoma F9 cells into primitive and visceral endoderm. 838 21

The immediate early (IE) genes of human cytomegalovirus (HCMV) are expressed in lymphocytes and are known to transactivate both viral and cellular promoters. The mechanism by which IE gene products of HCMV transactivate expression of the HLA A2 gene promoter in Jurkat cells, a T-lymphocyte cell line, was investigated. Transient expression assays were performed using plasmids containing the HLA A2 promoter-regulatory region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE genes. The upregulation of the HLA A2 promoter by HCVM IE gene products was shown not to be secondary to either interferon-gamma or -alpha. Previously described MHC class I regulatory or enhancer elements such as the interferon-stimulated response element (ISRE), NF-kappa B and H2TF1 binding sequences, and the interferon consensus sequence (ICS) were not required for transactivation of the A2 promoter. Rather, the only known regulatory elements in the HLA A2 promoter necessary for both basal expression and transactivation by HCVM IE gene products are the CCAAT box and TATA box motifs. These results support a model in which HCVM IE gene products act through the minimal HLA A2 promoter elements to increase gene expression.
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PMID:Only the HLA class I gene minimal promoter elements are required for transactivation by human cytomegalovirus immediate early genes. 838 27

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a protein with a predicted molecular weight of 51,540 presenting amino acid sequence homology with the immediate-early regulatory protein ICP27 of herpes simplex virus type 1. To investigate the regulatory properties of the ORF4 gene product, we performed a series of transient expression assays in Vero cells, using a plasmid expressing ORF4 as effector and several VZV genes and heterologous genes as targets. The VZV target plasmids contained promoter/regulatory regions from genes belonging to the three putative VZV kinetic classes fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target plasmids consisted of promoter/regulatory regions of human cytomegalovirus, Rous sarcoma virus, and human immunodeficiency virus type 1 fused to the reporter gene. These experiments demonstrated that the ORF4 gene product activated expression of ORF62 in a dose-dependent fashion but had no effect on the expression of the three other putative immediate-early genes (ORF4, ORF61, and ORF63). When various amounts of ORF4 were transfected in the presence of early gene promoters, dose-dependent transactivation was evidenced with the thymidine kinase gene (ORF36) and the major DNA-binding protein gene (ORF29) promoters; interestingly, little activity was detected with the promoter of the DNA polymerase gene (ORF28). No activation of late gene expression, represented by the glycoprotein I and glycoprotein II genes, was seen even over a wide range of concentrations of input ORF4 plasmid. Expression of pCMVCAT, pRSVCAT, and pHIVCAT was also stimulated by the ORF4 gene product. CAT mRNA analysis showed that activation of VZV target promoters occurs at the transcriptional and/or posttranscriptional level.
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PMID:Characterization of the regulatory functions of varicella-zoster virus open reading frame 4 gene product. 838 35

Scores of homeobox gene-encoded transcription factors are expressed in a definite spatiotemporal pattern during embryogenesis and regulate a series of as yet unidentified target genes to help coordinate the morphogenetic process. We have suggested that homeobox gene products modulate the expression of adhesion molecule genes and have shown in cotransfection experiments that the promoters for the neural cell adhesion molecule (N-CAM) and cytotactin/tenascin genes respond to cues from different homeobox-containing genes. In this study, we show that the HoxC6 (Hox-3.3)-encoded homeoprotein binds to a DNA sequence in the N-CAM promoter CCTAATTATTAA, designated homeodomain binding site I (HBS-I). To test whether HoxC6 regulated N-CAM promoter activity, we cotransfected the Long and Short reading frame variants of Xenopus HoxC6 (CMV-HoxC6-L and CMV-HoxC6-S) driven by the human cytomegalovirus (CMV) promoter together with a chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse N-CAM promoter (N-CAM-Pro-CAT). Cotransfection of NIH 3T3 cells with either of the CMV-HoxC6 expression vectors stimulated N-CAM promoter-driven CAT expression. A 47-bp region from the N-CAM promoter that included HBS-I and an adjacent potential HBS, HBS-II, conferred HoxC6 regulation on a simian virus 40 minimal promoter. HBS-I was sufficient for transactivation of the minimal promoter by CMV-HoxC6-S. However, transcriptional activation by CMV-HoxC6-L required both HBS-I and HBS-II, inasmuch as mutation of either HBS-I, HBS-II, or both motifs abolished the response. These studies suggest that HBS-I is a target site for binding and transcriptional control of the N-CAM promoter by homeoproteins, although accessory DNA sequences (such as HBS-II) may also be required. Together with previous studies, these results support the notion that N-CAM gene expression may be controlled by different combinations of homeoproteins that appear in a place-dependent manner during embryogenesis.
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PMID:Binding and transcriptional activation of the promoter for the neural cell adhesion molecule by HoxC6 (Hox-3.3). 839 70

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.
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PMID:Efficiency of introns from various origins in fish cells. 839 51

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J.A. Kerry, M.A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.
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PMID:Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection. 852 51

Neuronal migration in brain is followed by differentiation of committed neurons and simultaneous apoptosis of uncommitted preneuronal cells due to a limiting supply of trophic factors and nutrients. We have dissected differentiation and apoptosis by designing a simple in vitro model for this nutrient deprivation using engineered neuronal cell lines stably transfected with a promoterless segment (G-21) of the intronless human serotonin1A receptor (5-HT1A-R) gene. Despite the use of widely different heterologous promoters (cytomegalovirus and Rous sarcoma virus) for the stable expression of G-21, a dramatic increase in expression of the 5-HT(1A)-R (five- to 15-fold) and its mRNA was always observed during degeneration and apoptosis of nutrient-deprived neuronal cells. Involvement in this induction of a 170-bp 5'-end untranslated sequence (5'-UT) (tail end of the 500-bp natural promoter) of G-21 was confirmed by stable transfection of neuronal cells with an SV-40 promoter-driven construct harboring the 5'-UT and the reporter chloramphenicol acetyltransferase (CAT) cDNA. Presence of the 5'-UT resulted in a threefold increase in CAT expression during nutrient deprivation in randomly chosen clones. The induction was also observed in the endogenous 5-HT1A-R, expressed by embryonic day 16 mouse hippocampal neurons, subsequent to nutrient deprivation and onset of degeneration. A trophic role of the 5-HT1A-R has been suggested in earlier studies. Considering the example of protective heat shock proteins, which are induced during various types of stress, our results suggest that stressed neuronal cells undergoing degeneration and apoptosis synthesize increased levels of 5-HT1A-R as a final attempt to survive.
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PMID:Induction of the serotonin1A receptor in neuronal cells during prolonged stress and degeneration. 863 58

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.
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PMID:GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. 864 9

Human cytomegalovirus phosphoprotein pp65 is targeted to the cell nucleus immediately after infection. Deletion and point mutation analysis of the pp65 gene expressed in insect cells showed that two hydrophilic regions (HP1 and HP2) within the pp65 C-terminal 40% each harboured an independent nuclear localization signal (NLS); strong association to the nuclear stroma also requires the N-terminal domain. Either region, when fused to chloramphenicol acetyltransferase, localized the reporter protein to the nucleus in insect cells as well as in NIH 3T3 cells and human lung fibroblasts. In addition, HP1 was found to be the target of pp65 Ser/Thr phosphorylation in insect cells and a prokaryotically expressed HP1 was actively phosphorylated in vitro by casein kinase II, for which two site clusters map in HP1. These findings indicate that pp65 includes two NLSs, one of which has the potential to be modulated by phosphorylation.
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PMID:Human cytomegalovirus pp65 lower matrix phosphoprotein harbours two transplantable nuclear localization signals. 868

The Gfi-1 proto-oncogene encodes a zinc finger protein with six C2H2-type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. In this report, we show that Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/T)GCA, was defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site was confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oliginucleotides defined the relative importance of specific bases at the consensus binding site. Deletion of individual zinc fingers demonstrated that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites were detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, were repressed by Gfi-1, and the repression was abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes.
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PMID:Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor. 875


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