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 complete nucleotide sequence of a functional clone of the large polymerase (L) gene of bovine respiratory syncytial virus (BRSV) strain A51908 was determined by analysis of cloned cDNAs obtained from genomic and mRNAs. The BRSV L gene is 6573 nt in length and the derived polypeptide has 2162 aa. Alignment of the sequences of the BRSV L gene, and its encoded protein, with sequences of the L gene and protein of human respiratory syncytial virus strain A2 showed 77% identity at the nucleotide level and 84% identity at the amino acid level. By comparison, the L gene and protein of avian pneumovirus showed only 50% identity at the nucleotide level and 64% identity at the amino acid level. A minigenome was constructed to encode a BRSV vRNA analogue containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative BRSV transcription motifs and flanked by the BRSV genomic termini. Transfection of plasmids encoding the BRSV minigenome, nucleocapsid protein (N), phosphoprotein (P) and L protein, each under the control of T7 promoter, into cells infected with a vaccinia virus recombinant expressing the T7 RNA polymerase gave rise to CAT activity and progeny with the minigenome. This result indicates that the N, P and L proteins are necessary and sufficient for transcription and replication of the BRSV minigenome and are functional. Further, inclusion of small amounts of the M2 protein along with the N, P and L proteins greatly augmented minigenome transcription.
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PMID:Sequence analysis of a functional polymerase (L) gene of bovine respiratory syncytial virus: determination of minimal trans-acting requirements for RNA replication. 974 33

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication.
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PMID:Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virus-specific monocistronic minigenomes. 976 19

To investigate whether vaccinia virus (VV) can augment gene expression of human immunodeficiency virus type 1 (HIV-1), co-transfection experiments were carried out in which recombinant plasmids containing various portions of the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into cultured cells. A high level of enhancement in CAT activity directed by the HIV-1 LTRs containing the enhancer sequence was observed in cells infected with VV, as in the cells infected with type 1 herpes simplex virus (HSV-1). The sequence responsible for this augmentation of CAT activity was different from that recognized by HIV-1 Tat. These data clearly demonstrated that VV transactivates HIV-1 LTR through a mechanism distinct from that of activation by HIV-1 Tat.
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PMID:Activation of HIV-1 enhancer sequence by vaccinia virus. 1002 58

The nonreplicating vaccinia virus MVA/T7 RNA polymerase hybrid system was tested with Petri dish electroporation for ectopic gene expression in human umbilical vein endothelial cells (HUVECs). A range of voltages (150-450 V), pulse times (10-40 ms), DNA concentrations (0-20 microg/ml) and infection levels (0-15 multiplicities of infection) were tested for effects on T7 promoter-directed chloramphenicol acetyltransferase (CAT) activity after MVA/T7RP infection. MVA/T7RP-directed expression was transient and at least 10 000-fold in excess of nonviral, cytomegalovirus enhancer-directed expression. Use of a Petri dish electrode with the MVA/T7RP system showed increased viability compared with a cuvette electrode. Overexpression of interleukin-2 alpha subunit (IL2Ralpha) pro- tein followed by anti-IL2Ralpha-directed magnetic immunoaffinity cell sorting allowed isolation of the transfected population. The high fidelity of cellular sorting was shown by segregation of CAT activity in the IL2Ralpha-sorted population after transfection of T7 promoter-directed bicistronic IL2Ralpha/CAT DNA. Expression of a panel of proteins including the fluorophore green fluorescent protein as detected by fluorescence microscopy and p21cip1, p27kip1, pp60c-src, FGF-1, pRb, p107 and pRb2/p130 proteins was also achieved. Thus, use of the nonreplicating vaccinia virus/T7 RNA polymerase expression system with Petri dish electroporation is feasible for certain applications for the manipulation of HUVECs by gene transfer.
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PMID:Endothelial cell DNA transfer and expression using petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system. 1049 Jul 72

The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5' untranslated region (UTR) and IGR and downstream by the S 3' UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase.
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PMID:NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs. 1072 20

Synthetic T7-driven cDNA minigenomes containing the bacterial chloramphenicol acetyltransferase gene as a reporter were derived from the genome of two salmonid novirhabdoviruses, infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). We showed that an exogenous IHNV RNA minigenome transfected into fish cells could be rescued following IHNV infection as it was replicated, encapsidated and transcribed. When cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3), transfected with the plasmid carrying the IHNV minigenome (genomic- and antigenomic-sense) and superinfected with IHNV, rescue of the minigenome was more efficient. Heterologous VHSV/IHNV rescue experiments failed. Finally, when the IHNV N, P and L proteins were expressed from cDNAs in cells, the minigenome was also successfully rescued, indicating that the nucleocapsid proteins were biologically functional. These data represent the first example of rescue experiments for non-mammalian rhabdoviruses replicating at a low temperature.
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PMID:Rescue of synthetic salmonid rhabdovirus minigenomes. 1090 31

Infectious hematopoietic necrosis virus (IHNV) is a Novirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14 degrees C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains.
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PMID:Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes. 1107 23

A recombinant mesogenic NDV strain, Beaudette C, and an engineered recombinant NDV expressing an additional gene were generated entirely from cloned cDNAs. For this purpose, a full-length cDNA clone of the virus genome, represented in eight different subgenomic fragments, was assembled in a transcription plasmid between a T7 RNA polymerase promoter and a hepatitis delta virus ribozyme sequence. Infectious NDV could be generated in the cells infected with recombinant vaccinia virus, which expressed T7 RNA polymerase, by simultaneous expression of antigenome-sense NDV RNA from the full-length plasmid and NDV NP, P, and L proteins from cotransfected plasmids. Recombinant virus was then amplified and recovered, either after inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free eggs or after further passage in cell culture. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to that of the parental wild-type virus. By using this system, a recombinant NDV containing a foreign gene encoding chloramphenicol acetyltransferase (CAT) was generated. To do this, the CAT transcription cassette containing the CAT open reading frame, flanked by NDV gene start and gene end sequence motifs, was inserted into the region between the HN and L genes of the full-length cDNA. This construct was then used in the generation of a recombinant NDV expressing CAT protein. The CAT gene was maintained stably for at least eight passages without any detectable loss of the gene from the recombinant. Generation of the recombinant virus, however, was associated with reduced plaque size, slower replication kinetics, and more than 100-fold decrease in yield. In addition, the virus showed an increase in mean death time for eggs and a lower intracerebral pathogenicity index in day-old chicks, implicating attenuation of the recombinant virus. Thus, introduction of an additional gene into the NDV genome represents a method to achieve growth retardation and attenuation. These results also indicate that NDV can be engineered to express foreign protein stably and can be manipulated in the future for use as a vaccine vector.
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PMID:Recovery of a virulent strain of newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation. 1111 92

Tacaribe virus (TV), the prototype of the New World group of arenaviruses, comprises a single phylogenetic lineage together with four South American pathogenic producers of hemorrhagic disease. The TV genome consists of two single-stranded RNA segments called S and L. A reconstituted transcription-replication system based on plasmid-supplied TV-like RNAs and TV proteins was established. Plasmid expression was driven by T7 RNA polymerase supplied by a recombinant vaccinia virus. Plasmids were constructed to produce TV S segment analogs containing the negative-sense copy of chloramphenicol acetyltransferase (CAT) flanked at the 5' and 3' termini by sequences corresponding to those of the 5' and 3' noncoding regions of the S genome (minigenome) or the S antigenome (miniantigenome). In cells expressing N and L proteins, input minigenome or miniantigenome produced, respectively, encapsidated miniantigenome or minigenome which in turn produced progeny minigenome or progeny miniantigenome. Both minigenome and miniantigenome in the presence of N and L mediated transcription, which was analyzed as CAT expression. Coexpression of the small RING finger Z (p11) protein was highly inhibitory to both transcription and replication mediated by the minigenome or the miniantigenome. The effect depended on synthesis of Z protein rather than on plasmid or the RNA and was not ascribed to decreased amounts of plasmid-supplied template or proteins (N or L). N and L proteins were sufficient to support full-cycle RNA replication of a plasmid-supplied S genome analog in which CAT replaced the N gene. Replication of this RNA was also inhibited by Z expression.
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PMID:Transcription and RNA replication of tacaribe virus genome and antigenome analogs require N and L proteins: Z protein is an inhibitor of these processes. 1171 15

The paramyxovirus simian virus 5 (SV5) has seven genes but encodes eight known viral proteins. The V/P gene is transcribed into two mRNA species: V mRNA from a faithful transcription of the gene and P mRNA from transcription with addition of two G residues at a specific site of the gene. V, a 222-amino acid (AA) residue protein, and P, a 392 AA residue protein, share an identical N-terminus domain of 164 amino acid residues. P is essential for SV5 RNA replication and transcription. Whereas it is known that V plays important roles in virus pathogenesis, the role of V in SV5 replication and transcription is not clear. A mini-genome system, free of vaccinia virus gene expression system, consisting of plasmids expressing NP, P, and L, as well as a plasmid encoding a reporter gene, chloramphenicol acetyltransferase (CAT) flanked by SV5 trailer and leader sequences under control of a bacteriophage T7 RNA polymerase promoter, has been established to examine the role of V in SV5 RNA transcription and replication. Addition of V-expressing plasmid in the mini-genome system caused inhibition of the reporter gene expression, suggesting that V plays a role in regulating SV5 gene expression. By examining the amount of encapsidated viral RNA genome using reverse transcription with primer annealing to viral anti-genome RNA and PCR, it was found that expression of V reduced the amount of viral RNA genome in the mini-genome system, suggesting that V inhibits viral RNA replication. To examine whether the V protein inhibits viral RNA transcription as well, a mini-genome system with a defective anti-genome promoter (AGP) such that a reporter gene (luciferase, Luc) expression is only derived from transcription of newly produced mini-genome and not from de novo replicated viral genome due to the defect in replication element has been utilized. The V protein inhibited luciferase expression from the mini-genome with a defective AGP, suggesting V inhibits SV5 transcription. Thus, SV5 V inhibits both SV5 RNA replication and transcription.
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PMID:The role of simian virus 5 V protein on viral RNA synthesis. 1595 Sep 97


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