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)

RNA molecules were prepared that consisted of an mRNA encoding chloramphenicol acetyltransferase embedded within the sequence of midivariant RNA, which is a template for the RNA-directed RNA polymerase Q beta replicase. These recombinant RNAs were shown to be bifunctional: they are amplified exponentially by incubation with Q beta replicase, and the replicated RNA serves as template for the cell-free synthesis of enzymatically active chloramphenicol acetyltransferase. The availability of amplifiable mRNAs will enable relatively large amounts of protein to be synthesized in vitro.
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PMID:Amplifiable messenger RNA. 146 96

EBNA 1 is the only antigen expressed in both Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma cells. Previous studies showed that the mRNA of EBNA 1 in these two tumor tissues was initiated from a promoter located in the Bam HI F fragment (Fp) on the viral genome. Two regulatory elements located in the downstream Bam HI Q region include an EBNA 1 binding site and a positive regulatory region between the Fp and the EBNA1 binding site. This data strongly suggested that a cellular factor(s) may modulate the usage of the Fp. To locate the shortest responsible viral sequence, we constructed a series of luciferase gene and chloramphenicol acetyltransferase (CAT) gene plasmids that contained various portions of the Bam HI F/Q region. Plasmid DNA was then introduced into cells to examine the promoter activity of each construct. By this method, we identified a 186-bp fragment within the Bam HI Q region that possessed the highest activity. This promoter was designated as Qp and found to be orientation-dependent and down-regulated by EBNA 1 in both the type I BL cells and human epithelial cells. Furthermore, RNase protection assay showed that a transcription initiation site was located at nucleotide 62,416 of the EBV genome. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis further confirmed that the transcript was initiated from the Qp, and not the Fp. Therefore, our data suggested that a novel promoter, Qp, located within the Bam HI Q existed for the EBNA 1 expression in the latently infected type 1 BL cells. The biological significance of the selection of the Qp needs further investigation.
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PMID:Identification of a novel promoter located within the Bam HI Q region of the Epstein-Barr virus genome for the EBNA 1 gene. 766 54

Overlapping cDNAs representing the complete L segment of Rift Valley fever virus were assembled, and the L protein was expressed via a recombinant vaccinia virus. The transcriptase activity of the L protein was assayed with two types of templates: natural ribonucleoproteins (RNPs) and artificial genome-like RNAs. RNPs purified in a CsCl gradient did not retain the RNA polymerase function, but the activity was restored when the L cDNA was expressed in mammalian cells via a recombinant vaccinia virus. Indeed, after transfection of transcriptase-depleted RNPs in cells infected with the recombinant vaccinia virus expressing the L protein, the mRNAs coding for the N and NSs proteins and to a lesser extent, those coding for the glycoproteins were synthesized as well as the corresponding proteins. The transcriptase activity of the recombinant L protein was then investigated by using synthetic templates containing the reporter chloramphenicol acetyltransferase gene in the antisense orientation flanked by the 3' and 5' noncoding region of the S genomic segment. Our results indicate that after transfection of the RNA templates, transcription was achieved in cells coexpressing both the L and N proteins. Together, the experiments demonstrate that the two proteins N and L are absolutely required and sufficient to reconstitute the transcriptase activity.
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PMID:The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules. 776 55

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.
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PMID:Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. 878 Nov 19

A previous report described the recovery from cDNA of infectious recombinant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995). Here, the system was used to construct recombinant RSV containing an additional gene encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs, the transcription signals for the viral RNA-dependent RNA polymerase. The RSV-CAT chimeric transcription cassette was inserted into the region between the G and F genes of the complete cDNA-encoded positive-sense RSV antigenome, and infectious CAT-expressing recombinant RSV was recovered. Transcription of the inserted gene into the predicted subgenomic polyadenylated mRNA was demonstrated by Northern (RNA) blot hybridization analysis, and the encoded protein was detected by enzyme assay and by radioimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild-type recombinant RSV. Consistent with this finding, the CAT-containing and wild-type viruses were very similar with regard to the levels of synthesis of the major viral proteins. Each of 25 RSV isolates obtained by plaque purification following eight serial passages expressed CAT, showing that the foreign gene was faithfully maintained in functional form. Analysis by reverse transcription and PCR did not reveal evidence of deletion of the foreign sequence. This finding demonstrated that the RSV genome can accept and maintain an increase in length of 762 nucleotides of foreign sequence and can be engineered to encode an additional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in virus growth and 20-fold decrease in virus yield in vitro. Thus, introduction of an additional gene into the RSV genome might represent a method of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.
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PMID:Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene. 879 98

Nitrate increases the transcription of the two Arabidopsis thaliana nitrate reductase genes. We demonstrated previously that 238 and 330 bp of the 5' flanking regions, designated as NP1 and NP2, of the two nitrate reductase genes NR1 and NR2, respectively, are sufficient for nitrate-dependent transcription (Y. Lin, C.-F. Hwang, J.B. Brown, C.-L. Cheng [1994] Plant Physiol 106: 477-484). Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (LS) analysis. In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity. To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used to measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction. The single LS mutant in NP1 lost its response to nitrate, whereas the two LS mutants in NP2 partially lost their response to nitrate. A 12-bp sequence is conserved between the NP1 site and the two NP2 sites. This sequence motif is also conserved in the 5' flanking regions of other nitrate-inducible plant genes. Gel mobility shift experiments indicate that these three regions bind to similar proteins. The binding is constitutive with respect to nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves.
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PMID:Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes. 908 75

The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP. To determine if the N-terminal mutants could support the expression and replication of a model influenza B virus RNA, the genes encoding the subunits of the viral RNA-dependent RNA polymerase (PA, PB1, and PB2) were cloned. Coexpression of NP and the P proteins in 293 cells was found to permit the expression and replication of a transfected model RNA based on segment 4 of B/Maryland/59, in which the hemagglutinin-coding region was replaced by a chloramphenicol acetyltransferase gene. The expression and replication of the synthetic RNA were not affected by the replacement of NP with NP mutants lacking the N-terminal 51 or 69 residues, indicating that the N-terminal extension is not required for transcription or replication of the viral RNA. In addition, we report that the influenza B virus NP cannot be functionally replaced by type A virus NP in this system.
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PMID:The N-terminal extension of the influenza B virus nucleoprotein is not required for nuclear accumulation or the expression and replication of a model RNA. 957 10

A transcription system for Toscana virus (TOSV) (a member of the family BUNYAVIRIDAE:, genus PHLEBOVIRUS:) was constructed. For in vivo expression, the TOSV transcription system uses the viral N and L proteins and an S-like RNA genome containing the chloramphenicol acetyltransferase reporter gene in the antisense orientation flanked by the viral genomic 5'- and 3'-terminal S sequences. It was found that the N and L proteins represent the minimal protein requirement for an active transcription complex. To investigate the possibility of reassortment between TOSV and Rift Valley fever virus (RVFV), the activity of their polymerase complexes was tested on their heterologous S-like RNA genomes and this showed that both virus complexes were active. Moreover, hybrid transcriptase complexes with protein components originating from the two viruses were tested on both virus templates and only the combination RVFV L + TOSV N on RVFV S-like RNA was found to be active in this assay. These results suggest that virus reassortants might be generated whenever the two viruses infect the same host.
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PMID:Activity of Toscana and Rift Valley fever virus transcription complexes on heterologous templates. 1125 82

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences.
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PMID:Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites. 1156 76

We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the Bunyaviridae family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol. 75:1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5'- and 3'-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and 2 to 4; site B, positions 8 and 8, where underlined nucleotides refer to positions in the vRNA 3' end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5' and 3' ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5' and 3' ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.
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PMID:Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance. 1236 28


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