Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA

Oda, Kinichiro; Joklik, Wolfgang K.

doi:10.1016/0022-2836(67)90047-2

Cited by 255 publications

(91 citation statements)

References 22 publications

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“…The amount of gpl6O-cross-reactive 24-h indicated that the RNA was fairly stable. This is an intriguing result, since previous studies have suggested that in vaccinia virus-infected cells, both cellular and viral mRNAs have very short half-lives (13,17,18). It is our intention to further characterize the T7 transcripts, with particular regard to the ,cribed a simple and structure of their 5' and 3' ends.…”

mentioning

confidence: 74%

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

Fuerst¹,

Earl²,

Moss³

1987

Mol. Cell. Biol.

395

290

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A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli (-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system. Molecular cloning vectors have been developed that allow expression of heterologous genes in procaryotic and eucaryotic cells. Genetic elements carried by these vectors typically confer drug resistance, ability to replicate autonomously, and regulatory controlling elements juxtaposed to the target gene of interest. While this approach has been extensively used in bacterial and mammalian cell expression systems, certain features of one system may be advantageous over another. Bacteria are easy to use and high expression is often attainable, but synthesis of native eucaryotic proteins is frequently not achieved. Mammalian cells can faithfully express eucaryotic proteins, but usually at relatively low levels. An expression system that used procaryotic transcriptional elements in mammalian cells might have important advantages since the catalytic activity, induciblity, and promoter specificity are well characterized.For these reasons, a chimeric expression system that used favorable transcriptional components from bacteria in a eucaryotic milieu may offer a highly specific and efficient method for the synthesis of ecuaryotic proteins.A eucaryotic transient-expression system based on a recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase in the cytoplasm of infected cells was recently described (5). Plasmids containing the target genes flanked by T7 promoter and termination sequences were introduced into infected cells by transfection procedures. The efficiency of the vaccinia virus-T7 transient system relative to that of more conventional mammalian transient expression systems was attributed to the high catalytic activity of T7 RNA polymerase and stringent T7 promoter specificity. Since it is possible to infect tissue culture cells synchronously with vaccinia virus, the absolute level of expression was probably limited by transfection of the target gene. It would seem that the efficiency of the system would be enhanced if vaccinia virus were used...

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mentioning

confidence: 74%

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

Fuerst¹,

Earl²,

Moss³

1987

Mol. Cell. Biol.

395

290

View full text Add to dashboard Cite

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“…Approximately half of the vaccinia virus genes belong to the early class (Oda & Joklik, 1967). A single early promoter, that of the 7?5K gene, has been characterized in detail (Davison & Moss, 1989b).…”

Section: Early Gene Transcriptionmentioning

confidence: 99%

Vaccinia virus transcription

Broyles

2003

Journal of General Virology

194

168

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“…Polysomal RNA was extracted using a chloroform/ isoamyl alcohol mixture [39] and poly(A)containing mRNA was purified by affinity chromatography on oligo(dT)-cellulose according to Aviv and Leder [40].…”

Section: Preparation Of Polysomes and M R N A From Rat Livermentioning

confidence: 99%

Studies on the Biosynthesis of Microsomal Membrane Proteins

Okada

Frey

Guenthner

et al. 1982

European Journal of Biochemistry

113

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The site of synthesis and mechanism of insertion into membranes of several microsomal polypeptides was studied using translation system programmed in vitro with polysomes or with mRNA extracted from free and membrane-bound rat liver polysomes. Primary translation products of cytochrome bs, NADH : cytochrome b5 oxidoreductase, NADPH : cytochrome P-450 oxidoreductase and epoxide hydrolase were isolated by specific immunoprecipitation and compared with the mature proteins. The following observations were made :1. While cytochrome b5 and NADH : cytochrome bs oxidoreductase are synthesized in free polysomes, NADPH : cytochrome P-450 oxidoreductase and epoxide hydrolase are made in membrane-bound polysomes.2. In all cases the radioactively labelled products synthesized in vitro comigrated with the purified native polypeptides. Since these proteins are not glycoproteins, it can be inferred that in vivo they do not undergo proteolytic cleavage during or after translation.3. Levels of translatable mRNA coding for NADPH : cytochrome P-450 oxidoreductase or epoxide hydrolase were increased by phenobarbital or by various carcinogens respectively. 4. A comparison of amino-terminal segments of the native epoxide hydrolase and that synthesized in vitro showed that this protein retains an amino-terminal polypeptide sequence which resembles the transient insertion signal found in presecretory proteins.5. The amino-terminal region of NADPH : cytochrome P-450 oxidoreductase contains 7 leucine residues in its first 15 amino acids and can, therefore, be presumed to be the hydrophobic portion which anchors this polypeptide to the membrane.Mechanisms which may account for the spatial disposition of the membrane proteins and for their selective accumulation in endoplasmic reticulum membranes are discussed.There is now considerable interest in the mechanisms which serve to ensure the specific incorporation of newly synthesized polypeptides into the different organellar membranes which characterize the eukaryotic cell. The endoplasmic reticulum plays an important role in the biogenesis of membranes by providing sites for cotranslational insertion of a variety of polypeptides, which are synthesized by membrane-bound ribosomes and are subsequently transferred to other subcellar compartments (cf. [l]).In the hepatocyte and in many other cell types the endoplasmic reticulum constitutes the most extensive membrane system of the cell and contains membrane-associated enzymes which play an important role in metabolic and biosynthetic processes affecting a variety of substrates. The biosynthesis of integral membrane proteins which are characteristic of the endoplasmic reticulum raises interesting questions concerning the incorporation of the polypeptides into the membranes and the means which allow for their retention in the endoplasmic reticulum by distinguishing between them and those to be transferred to other membrane systems [2].To study these questions we have selected several proteins of the hepatocyte endoplasmic reticulum membranes which...

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Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA

Cited by 255 publications

References 22 publications

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

Vaccinia virus transcription

Studies on the Biosynthesis of Microsomal Membrane Proteins

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