Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have shown that the molecules of P protein associated with transcriptionally active Sendai virus nucleocapsids are arranged in discrete clusters. Our study investigates whether or not this localized distribution is due to the existence of only a few P protein binding sites on the nucleocapsid core. We used immunoelectron microscopy to examine whether additional P proteins could bind at locations between the groups of endogenous P proteins. To differentiate between endogenous and added proteins, we constructed a recombinant gene which instructs the in vitro synthesis of a chimeric protein containing the carboxyl-terminal nucleocapsid-binding region of P protein, fused to chloramphenicol acetyltransferase (CAT). Immunogold labelling, using an antibody to the CAT moiety, revealed at the electron microscope level, that the chimeric product bound to nucleocapsids at many sites located over the entire length of the nucleocapsid. This indicated that the localized distribution of P protein molecules is not due to a limited number of P protein binding sites on the nucleocapsid core.
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PMID:Localization of P protein binding sites on the Sendai virus nucleocapsid. 215 9

Members of the Paramyxoviridae family utilize a variety of different strategies to increase coding capacity within their P cistrons. Translation initiation at alternative 5'-proximal AUG codons is used by measles virus (MV) to express the virus-specific P and C proteins from overlapping reading frames on their mRNAs. Additional species of mRNAs are transcribed from the MV P cistron by the insertion of extra nontemplated G residues at a specific site within the P transcript. Addition of only a single nontemplated G residue results in the expression of the V protein, which contains a unique carboxyl terminus. We have used an Escherichia coli system to express MV P cistron-related mRNAs and proteins. We have found that ribosomal frameshifting on the MV P protein mRNA is capable of generating a previously unrecognized P cistron-encoded protein that we have designated R. Some ribosomes which have initiated translation of the P protein mRNA use the sequence TCC CCG AG (24 nucleotides upstream of the V protein stop codon) to slip into the -1 reading frame, thus translating the sequence as TC CCC GAG. The resulting R protein terminates five codons downstream of the frameshift site at the V protein stop codon. We have gone on to use a chloramphenicol acetyltransferase reporter system to demonstrate that this MV-specific sequence is capable of directing frameshifting during in vivo translation in eukaryotic cells. Analysis of immunoprecipitated proteins from MV-infected cells by two-dimensional gel electrophoresis allowed detection of a protein species consistent with R protein in MV-infected cells. Quantitation of this protein species allowed a rough estimation of frameshift frequency of approximately 1.8%. Significant stimulation of ribosomal frameshift frequency at this locus of the MV P mRNA was mediated by a downstream stimulator element which, although not yet fully defined, appeared to be neither a conventional stem-loop nor an RNA pseudoknot structure.
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PMID:Ribosomal frameshifting during translation of measles virus P protein mRNA is capable of directing synthesis of a unique protein. 747 85

Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle of the virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.
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PMID:Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system. 823 1

Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.
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PMID:Construction of a Sonchus Yellow Net Virus minireplicon: a step toward reverse genetic analysis of plant negative-strand RNA viruses. 2388 70