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 gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.
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PMID:Regulation of the Epstein-Barr virus DNA polymerase gene. 131 4

We have been studying the role of human cytomegalovirus (HCMV) as a potential cofactor in human immunodeficiency virus (HIV)-related disease. The clinical relevance of HCMV is highlighted by the fact that it is a principal viral pathogen in patients with AIDS and is known to infect the same cells as HIV. In this study, we focused on the molecular interactions between HIV and HCMV in human fibroblasts and in the human glioblastoma/astrocytoma-derived cell line U373 MG, cells which can be productively infected by both viruses. Because these cells are CD4-, we used HIV pseudotyped with a murine amphotropic retrovirus as described previously (D. H. Spector, E. Wade, D. A. Wright, V. Koval, C. Clark, D. Jaquish, and S. A. Spector, J. Virol. 64:2298-2308, 1990). Initial studies showed that when cells were preinfected with HIV (Ampho-1B) for 5 days and then superinfected with HCMV, HIV antigen production dropped significantly in the coinfected cells but continued to rise in cells infected with HIV (Ampho-1B) alone. HCMV production, however, was unaffected by the presence of HIV. Further analysis showed that HIV steady-state RNA levels and gag and env protein production were also inhibited in the presence of HCMV. The transcriptional inhibition of HIV was particularly surprising in view of the previous results of several other laboratories as well as our own that HCMV infection stimulates HIV long terminal repeat-chloramphenicol acetyltransferase (LTR-CAT) expression in transient expression assays. To investigate this further, we transfected the HIV LTR-CAT construct into either uninfected cells or cells which had been preinfected with HIV. The cells were infected with HCMV 24 h posttransfection and assayed for CAT gene expression at 48 h after HCMV infection. Although there was some stimulation of the LTR-CAT in cells that were dually infected by HIV and HCMV, it was 16-fold less than that in the cells infected only with HCMV. This suggests that in the presence of the HIV infection, the stimulation of the HIV LTR-CAT gene by HCMV is significantly reduced. Experiments with UV-irradiated HCMV and the HCMV DNA polymerase inhibitor ganciclovir showed that HCMV transcription is necessary for the reduction in HIV production to occur; however, replication of the HCMV genome or any events which take place after DNA replication are not necessary. These results, coupled with the observation that inhibition is usually first seen between 8 and 24 h after HCMV infection, suggest that an HCMV early protein is involved in repression of HIV.
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PMID:Human cytomegalovirus inhibits human immunodeficiency virus replication in cells productively infected by both viruses. 165 86

Several cis-acting transcriptional silencer elements that are dispersed over a long (more than 1 kb) region upstream of the mouse DNA polymerase-beta gene repress transcription not only of this gene but also of heterologous promoter-enhancers of other genes. The effect of DNA replication on the functions of these silencer elements was examined by combining them with transiently replicating plasmids carrying the chloramphenicol acetyltransferase (CAT) gene and the replication origins (ori) of polyoma virus and SV40 DNA. The silencers functioned efficiently when these plasmids were transfected into mouse NIH 3T3 and monkey CV1 cells. These cells lack T antigen and therefore do not allow replication of the plasmids. The silencers also functioned when the plasmids contained nonfunctional SV40 ori. In contrast, the negative effect of the silencer was not observed when the plasmids were transfected into cells producing large T antigens, such as MOP8 cells and COS1 cells. The observed differences in the silencer function between the permissive and nonpermissive cells was not due to differences in plasmid copy numbers in the cells. The results indicate that ongoing DNA replication can completely overcome the negative effects of the silencer elements on transcription.
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PMID:DNA replication can overcome the silencer function on transcription. 228 8

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.
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PMID:The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome. 258 Dec 53

The polA gene of Streptococcus pneumoniae cloned in the recombinant plasmid pSM22 is expressed in Bacillus subtilis. Extracts of B. subtilis polA mutants containing pSM22 showed 6 times more DNA polymerase activity than extracts of wild-type cells without the plasmid. Complete complementation of the B. subtilis polA5 and polA59 mutations with respect to in vivo resistance to UV irradiation and methyl methanesulfonate was observed when four copies of the pneumococcal polA gene were present in each cell. Ectopic integration of the polA gene together with a cat marker into the chromosome of B. subtilis gave chromosomal insertions containing single and double doses of the pneumococcal polA gene. Correlation with gene dosage was observed for both chloramphenicol acetyltransferase and DNA polymerase activities measured in vitro. Depending on the number of copies of the S. pneumoniae polA gene present, restoration of DNA repair functions in polA mutants of B. subtilis was either partial or complete.
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PMID:Complementation of Bacillus subtilis polA mutants by DNA polymerase I from Streptococcus pneumoniae. 312 7

We isolated and delimitated the Drosophila ras2 promoter region, determined its sequence and mapped the transcription units expressed in this region. The results showed that the Drosophila ras2 gene is flanked by another transcription unit, which codes for two larger transcripts, 2.5 and 2.9 kb long. Orientation experiments, in which sense and antisense RNA probes were used, revealed that both these and the ras2 transcripts are synthesized from different DNA strands. Thus, the flanking transcription unit is in the opposite polarity relative to the ras2 gene. The transcription start sites of the ras2 gene and the flanking transcription unit were determined by external primer extension with T4 DNA polymerase and by RNAase-protection assay and were found to be only 94 nucleotides apart. Apparently, the Drosophila ras2 promoter is a bidirectional promoter. Nucleotide sequence analysis revealed that the 5'-end of the ras2 transcript is within an inverted repeat of the insect cap box. TATA- and GC-like boxes were also found. Analysis of direct and inverted repeats in the promoter region suggested that it is asymmetrical. To demonstrate promoter activity, each side of the ras2 bidirectional promoter was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and tested by transfecting Drosophila Schneider 2 culture cells. Significant CAT activity was obtained with both transcription fusions.
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PMID:A bidirectional promoter is regulating the Drosophila ras2 gene. 341 73

A segment from the pre-s region of the woodchuck hepatitis virus (WHV) was inserted into an open reading frame vector allowing for the expression in Escherichia coli of viral determinants as part of a fusion protein. The bacterially synthesized fusion molecule contained eight amino acids from beta-galactosidase (beta-gal) at the N terminus, followed by 89 pre-s-encoded amino acids and 219 amino acids of chloramphenicol acetyltransferase (CAT) at the C terminus (beta-gal:pre-s:CAT). This tribrid protein was used to generate antiserum which had a significant titer to the viral portion of the fusion polypeptide. Anti-beta-gal:pre-s:CAT was used in Western blot analysis to identify viral proteins containing pre-s-encoded determinants. Antiserum to the tribrid molecule recognized four WHV polypeptides with molecular masses of 33, 36, 45, and 47 kilodaltons, each of which was also recognized by a monoclonal antibody to WHV surface antigen. Using the same anti-tribrid serum, we also identified analogous polypeptides from ground squirrel hepatitis virus. The antiserum was also used to immunoprecipitate virus particles containing endogenous DNA polymerase activity, indicating that pre-s determinants are found on the surface of mature virions. Based on previous computer studies and the location of pre-s-encoded molecules on the surface of virus particles, a role in hepadnavirus host cell entry is suggested for these polypeptides.
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PMID:Identification and localization of pre-s-encoded polypeptides from woodchuck and ground squirrel hepatitis viruses. 394 37

Plasmid constructs containing the 1.2-kb RNA promoter from the long terminal repeat region of human cytomegalovirus (HCMV) display the early-phase regulation of this promoter but lack the characteristic late induction (E. J. Wade, K. M. Klucher, and D. H. Spector, J. Virol. 66:2407-2417, 1992). To determine if the HCMV origin of replication (oriLyt) was necessary and sufficient for the late induction of the 1.2-kb RNA promoter, we cloned a 9.6-kbp segment of the origin of replication onto the p456 OCAT plasmid containing the 1.2-kb RNA promoter. This plasmid was designated ori456 OCAT. A control construct, which contains all of the same sequences as the ori456 OCAT construct except that a 2.4-kbp segment derived from HCMV EcoRI segment U is inverted in orientation to disrupt the origin function, was designated inv456 OCAT. After electroporation into human fibroblast cells and infection with HCMV 24 h later, ori456 OCAT replicated and showed the same early and late transcription pattern as the authentic viral 1.2-kb RNA. Under similar conditions, the inv456 OCAT neither replicated nor showed late induction. Experiments using plasmids synthesized in bacteria lacking methylation activity demonstrated that the late induction was not dependent on the change in methylation state of the plasmids. Ganciclovir, an inhibitor of the HCMV DNA polymerase, was used to demonstrate the replication dependence of the expression of the virally encoded 1.2-kb RNA, while the nearby early 2.7-kb RNA was unaffected. Ganciclovir also inhibited the late induction of the chloramphenicol acetyltransferase gene from ori456 OCAT, while expression from inv456 OCAT increased. Site-specific mutations in two previously identified important regulatory elements of the 1.2-kb RNA promoter, the AP1-binding site and the CATA site, indicated that these sites continue to contribute to promoter activity at late times but that the replication-dependent late induction acts independently of these sites. Possible mechanisms underlying the late induction are discussed.
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PMID:The human cytomegalovirus origin of DNA replication (oriLyt) is the critical cis-acting sequence regulating replication-dependent late induction of the viral 1.2-kilobase RNA promoter. 808 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

Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when appended to a heterologous basal promoter, is highly responsive to HHV-6 infection. Two protein complexes were found to bind in a specific manner to the ATF/CREB motif in both uninfected and HHV-6-infected T-cell nuclear extracts. Site-specific mutation of the ATF/CREB site resulted in loss of protein binding as well as loss of promoter activity in HHV-6-infected cells.
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PMID:An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter. 815 67


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