Top PDF Protein Synthesis in Cell-Free Systems: an Effect of Interferon

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

Stimulation of amino acid incorporation by EMC RNA with untreated "74S" ribosomes from control chick embryo fibroblasts CEF, U and CEF which had been exposed to 35 0 and 140 A units of i[r]

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Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNAF

Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNAF

Characterization of the polypeptide products synthesized in response to EMC RNA with time in the cell-free systems in the presence of cell sap fractions from vaccinia virus-infected and [r]

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Activation of the beta interferon promoter by paramyxoviruses in the absence of virus protein synthesis

Activation of the beta interferon promoter by paramyxoviruses in the absence of virus protein synthesis

and the IFN-b promoter in the presence of protein synthesis inhibitors. We investigated next whether preparations of paramyxoviruses that were generated by low-multiplicity passage, and therefore were not deliberately enriched for DI viruses (i.e. our working stocks of virus), could activate the IFN-induction cascade in the absence of virus protein synthesis. IRF3 activation could be detected in untreated cells infected with our vM0 preparations of PIV5-VDC and SeV, but, in contrast to infections with DI-rich preparations of these viruses, treatment with CHX had an inhibitory effect on IRF3 activation in infected cells (Fig. 4a, b). For these virus preparations, therefore, inhibition of virus protein synthesis limited activation of the IFN-induction cascade. This inhibition was not complete, however, and small amounts of p-IRF3 were detectable even in the presence of CHX. This low-level activation of the IFN- induction cascade by these virus stocks in the absence of protein synthesis suggests that DIs are present even in virus preparations that have been generated so as to minimize DI generation. Our working stock of MuV (termed MuV cl.3) was generated by plaque purification of our DI-rich MuV preparation (MuV bulk) [for further characterization, see Chen et al. (2010)] and could therefore be considered ‘DI- poor’; consistent with this, IRF3 phosphorylation was not detectable in MuV cl. 3-infected cells in the presence of CHX (Fig. 4c). Replication was required for IRF3 activation by this virus preparation, as p-IRF3 was detectable in untreated but not CHX-treated cells.
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Mengovirus leader is involved in the inhibition of host cell protein synthesis.

Mengovirus leader is involved in the inhibition of host cell protein synthesis.

cade of phosphorylation and dephosphorylation reactions and will finally result in the transcription of specific genes (14). The process of signal transduction and subsequent transcription triggered by interferon can be influenced by kinase inhibitors. The kinase inhibitor 2-aminopurine has been described as a selective inhibitor of gene expression induced by interferons and double-stranded RNA (18). However, it was also sug- gested that 2-aminopurine may interfere more generally with host cell transcription or translation (23). Hence, 2-aminopu- rine may be able to complement the mutant virus with respect to the inhibition of host cell protein synthesis. Treatment of L929 cells with 2-aminopurine and subsequent infection with the mengovirus L deletion mutant indeed resulted in mutant virus production increasing from 1 to approximately 60% of wild-type yield. Analysis of protein synthesis showed that, after treatment of L929 cells with 2-aminopurine, host cell transla- tion was completely inhibited after mutant virus infection whereas viral translation was not affected. In a study of the function of poliovirus 2A, a viable mutant which did not cleave eIF-4G was constructed (10). Similar to the mengovirus L deletion mutant, this 2A mutant was incapable of inhibiting host cell translation completely and viral protein synthesis was reduced. As for the effect of 2-aminopurine in L deletion mutant mengovirus-infected L929 cells, treatment of cells with 2-aminopurine before infection with the poliovirus 2A mutant resulted in the rescue of poliovirus protein synthesis and in the complete host cell translation shutoff (10).
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Virus Protein Synthesis in Animal Cell-Free Systems: Nature of the Products Synthesized in Response to Ribonucleic Acid of Encephalomyocarditis Virus

Virus Protein Synthesis in Animal Cell-Free Systems: Nature of the Products Synthesized in Response to Ribonucleic Acid of Encephalomyocarditis Virus

The autoradiographs shown are from analyses of the following systems: A and B, untreated Krebs cell microsomes assayed with Krebs cell sap and 35S-methionine in the absence A and presenc[r]

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Cell-free expression systems for the construction of artificial cells

Cell-free expression systems for the construction of artificial cells

The RNA polymerases were tested for transcription activity and compared to their commercial counterparts. To monitor RNA traces in real time, aptamers were used that bind fluorophores enhancing their quantum yield. T7 RNA polymerase performed better than commercial polymerase when the same amount was used for transcription, meaning that the purified stock was characterized by a higher U/µL count. E. coli RNA polymerase core enzyme and σ 70 transcription factor were mixed in an in vitro transcription reaction to reconstitute the RNA polymerase holoenzyme. After testing different ratios between the two purified components, the optimal reaction concentrations observed were 126 nM and 311 nM for the core enzyme and the σ 70 transcription factor, respectively. These concentrations are consistent with the fact that specific and efficient RNA synthesis from bacterial and phage promoters is achieved when the core enzyme is saturated with σ 70 transcription factor. Purified E. coli RNA polymerase holoenzyme was performing better than the commercial holoenzyme. When testing in vitro transcription with E. coli RNA polymerase holoenzyme, the pRNA scaffold influence on malachite green aptamer activity was assessed. The transcription product of pRNA is reported to fold into a secondary structure that increases the stability and folding of up to three aptamers inserted into its loops. Malachite green aptamer was cloned into one of the pRNA loops and this setting was compared to the simple malachite green aptamer. Results show that pRNA is actually enhancing the folding of malachite green aptamer even if the effect is observed only when considerable amounts of RNA are produced. When the homemade PURE system was assembled, only T7 RNA polymerase transcription activity was observed. Translation of reporter fluorescent protein was not detectable. This was probably due to one or more inactive translation factor and/or tRNA synthetase.
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Brefeldin A Inhibits Cell-Free, De Novo Synthesis of Poliovirus

Brefeldin A Inhibits Cell-Free, De Novo Synthesis of Poliovirus

Paradoxically, there appears to be a requirement for some aspects of secretory function in the replication of poliovirus. This has been inferred by the inhibition of poliovirus replica- tion by the fungal metabolite brefeldin A (BFA) (20, 30, 53), a drug usually characterized as an inhibitor of membrane traffic in the normal cells. BFA is a potent inhibitor of viral RNA synthesis in the infected cell, but not of entry, translation, or morphogenesis (20, 30, 53). This effect is dependent on a host- cell function, since replication proceeds normally in resistant cell lines, and attempts to isolate resistant poliovirus mutants have failed (11). The best-studied effect of BFA on the normal cell is the inhibition of vesicle-dependent transport at various stages in the secretory pathway, destroying the native functions of the Golgi complex, endosomes, and lysosomes (19, 25, 27, 57). BFA was ultimately shown to prevent the activation of some members of the ADP-ribosylation factor (ARF) family, a group of polypeptides that are key regulators in the synthesis of secretory transport vesicles (41). ARFs comprise a family of small (21-kDa) GTP-binding cytosolic proteins that localize to membranes when bound to GTP. This event prompts the re- cruitment and assembly of a protein coat (composed of either the coatomer or clathrin complexes), thereby deforming the underlying membrane into a budding vesicle (14, 44, 56) which eventually results in the release of a coated vesicle. Uncoating occurs upon GTP hydrolysis (50), which requires an additional factor (29). Binding of ARF to guanosine 59-O-(3-thiotriphos- phate) (GTPgS), a nonhydrolyzable GTP analog, results in the accumulation of coated buds and vesicles (50). When hydro- lysis of GTP occurs, the coat components and ARF-GDP become cytosolic, but they can be recycled by a guanosine exchange factor (ARF-GEF), resulting in new ARF-GTP that renew the budding process. BFA inhibits the exchange step (although the effect on ARF-GEF may not be direct) (13, 18), effectively segregating ARF to the cytosol and preventing ves-
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Cell-free soluble expression of the membrane protein PsbS

Cell-free soluble expression of the membrane protein PsbS

9 are  both  taken  from  samples  where  PsbS  is  solubilized  in   β -­‐DM.  The  observed  differences  could   originate  from  the  presence  of  lipids  from  the  E.  coli  host  system  in  the  E.  coli  produced  PsbS  as   NMR   spectra   show   the   presence   of   protein-­‐associated   lipids   that   are   purified   along   with   the   protein  (data  not  shown).  We  suspect  that  lipids  mediate  the  refolding  of  PsbS  and  influence  the   conformations  of  the  non-­‐helical  contents.  The  influence  of  lipids  on  the  refolding  of  membrane   proteins   is   an   interesting   aspect,   which   is   not   easily   controlled   in   recombinant   expression   systems   using   host   cells,   but   which   could   be   further   explored   in   CF   synthesis,   where   selective   lipids  can  be  added  to  the  synthesis  reaction  or  during  the  subsequent  refolding  steps.    
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Stimulation of Poliovirus Synthesis in a HeLa Cell-Free In Vitro Translation-RNA Replication System by Viral Protein 3CDpro

Stimulation of Poliovirus Synthesis in a HeLa Cell-Free In Vitro Translation-RNA Replication System by Viral Protein 3CDpro

vitro reactions has no effect on the translation of viral proteins from the input RNA and on the processing of the polyprotein, we conclude that the enhancing activity of this protein is in- volved at a later stage of the viral growth cycle, such as RNA replication, encapsidation, or both. It should be noted, how- ever, that the protein has to be added to the reactions during the first 2 to 4 h postincubation, which is the time of optimal translation, to retain its maximal stimulatory activity. In addi- tion, after the replication complexes have been formed from the newly made viral proteins in the presence of 2 mM guani- dine HCl, 3CD pro loses its ability to stimulate virus production.
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F-Factor-mediated restriction of bacteriophage T7: protein synthesis in cell-free systems from T7-infected Escherichia coli F- and F+ cells.

F-Factor-mediated restriction of bacteriophage T7: protein synthesis in cell-free systems from T7-infected Escherichia coli F- and F+ cells.

To determine whether the translational defect in T7-infected F+ cells is due to a T7 late mRNA-specific translational block, or to a general decrease of F+ cell translational activity, w[r]

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Synthesis and cell free cloning of DNA libraries using programmable microfluidics

Synthesis and cell free cloning of DNA libraries using programmable microfluidics

We used M-CAD to generate a library of 22 rationally designed variants of the Azurine gene by programming a DMF LH scheme that enabled the parallel assembly and individual retrieval of explicitly specified gene vari- ants from the microfluidic cartridge. Assembly was accom- plished through binary overlap extension reactions between PCR products. To design an appropriate DNA library con- struction scheme and the corresponding oligonucleotides used in the construction process we performed a shared component computational analysis of the Azurine library using a heuristic for maximizing DNA reuse in DNA li- brary construction (35) and computational oligonucleotide design tools (9,32,36). These were also used for automat- ically generating a construction plan for the same library using a Tecan liquid handling robot controlled by a high level programming language (9). We translated this plan into two protocols for the construction of the library, one using liquid handling robots and the other using M-CAD (see Video S2 for simulation) and executed both. The M- CAD Azurine library construction process (Video S2) fol- lowed the aforementioned plan and consisted of PCR am- plification of building blocks from three different plasmid templates using 6 different primers (Supplementary Fig- ure S9), generating 12 PCR products (Supplementary Fig- ure S9, blue nodes a and b). These 12 PCR products were further assembled in a combinatorial fashion using binary overlap extension to yield 24 out of the 27 possible target combinations and a final PCR amplification of the 24 full length assembled targets using external primers was also performed (see Video S3). All 24 library targets were then individually eluted from the cartridge, sized using gel elec- trophoresis following off-cartridge amplification (Supple- mentary Figure S10) and DNA sequenced to verify their sequence. Their sequence was identical to the same con- structs made using a control construction of the same target molecules using liquid handling robot program (see supple- mentary sequencing file) and construction methodologies (27). To demonstrate that the method produced functional variants we analyzed one of the variants effect on the post- transcriptional control by an RNA binding protein. This was achieved by monitoring the production of the variant azurin protein in bacterial extracts by western blot analy- sis (Supplementary Figure S10). It shows that the mutant tested (which had relatively minor, synonymous alterations to an RNA binding site on the azurin mRNA coding re- gion) was functional and had substantially altered azurin protein expression.
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The influence of the penetrating cryoprotectors on protein synthesis in the cell-free system of the rat liver

The influence of the penetrating cryoprotectors on protein synthesis in the cell-free system of the rat liver

tivity of postmitochondrial supernantant of the rat liver as well as on aminoacylation processes has been investigated. The addition of the penetrating cryoprotectors – ethylene glycol and DMSO – re­ sulted in the concentration­dependant reversible inhibition of the protein biosynthesis and ami­ noacylation reaction in the cell­free system. These cryoprotectors at low concentrations intensified the stimulating effect of Mg 2+ on the cumulative

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Formation of Protease-Resistant Prion Protein in Cell-Free Systems

Formation of Protease-Resistant Prion Protein in Cell-Free Systems

The central proposal of most protein-only models for the infectious agent of the TSEs is that the putative infectious protein, i.e. PrP-res, directly interacts with its normal, host encoded homolog, PrP-sen, to convert it to PrP-res. In this way, it could propagate itself in the host without mediation by an agent specific nucleic acid (11-15). This fundamental ability of PrP-res to induce the conversion of PrP-sen to PrP-res (converting activity) was first demonstrated by mixing PrP-res purified from scrapie-infected brain tissue with immunoprecipitated 35S-PrP-sen and observing that 35S-PrP-sen was then transformed into 35S-PrP-res (16). This conversion was not observed in the absence of PrP- res or in the presence of another type of amyloid (Alzheimer’s beta). Furthermore, other labelled proteins were not converted to protease K (PK)- resistant forms in the reaction with PrP-res. Thus, the conversion reaction is PrP-specific and PrP-res dependent. The converting activity depends upon the unique conformational structure of PrP-res because, although partial, reversible unfolding of PrP-res stimulates the conversion efficiency, more complete irreversible denaturation eliminates the converting activity (16,17). Further analyses of the effect of denaturants on the converting activity of PrP-res have indicated that maintenance of the native folding of a C- terminal domain (~16 kDa in the aglycosyl structure) is important to allow refolding and recovery of converting activity upon dilution of the denaturant (17). The denaturation of this critical C-terminal domain coincided with large reductions in both converting activity and scrapie infectivity (18).
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AFG3L2 supports mitochondrial protein synthesis and Purkinje cell survival

AFG3L2 supports mitochondrial protein synthesis and Purkinje cell survival

Mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia type 28 and spastic ataxia-neurop- athy syndrome in humans; however, the pathogenic mechanism is still unclear. AFG3L2 encodes a subunit of the mitochondrial m-AAA protease, previously implicated in quality control of misfolded inner mitochondrial membrane proteins and in regulatory functions via processing of specific substrates. Here, we used a condi- tional Afg3l2 mouse model that allows restricted deletion of the gene in Purkinje cells (PCs) to shed light on the pathogenic cascade in the neurons mainly affected in the human diseases. We demonstrate a cell-autonomous requirement of AFG3L2 for survival of PCs. Examination of PCs prior to neurodegeneration revealed fragmen- tation and altered distribution of mitochondria in the dendritic tree, indicating that abnormal mitochondrial dynamics is an early event in the pathogenic process. Moreover, PCs displayed features pointing to defects in mitochondrially encoded respiratory chain subunits at early stages. To unravel the underlying mechanism, we examined a constitutive knockout of Afg3l2, which revealed a decreased rate of mitochondrial protein syn- thesis associated with impaired mitochondrial ribosome assembly. We therefore propose that defective mito- chondrial protein synthesis, leading to early-onset fragmentation of the mitochondrial network, is a central causative factor in AFG3L2-related neurodegeneration.
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Viral protein synthesis in Friend erythroleukemia cell lines.

Viral protein synthesis in Friend erythroleukemia cell lines.

The cells were propagated in Dulbecco-modified Eagle me328 Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest Viral protein synthesis was studied in two Friend virus-induc[r]

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The synthesis of polynucleotides for interferon induction

The synthesis of polynucleotides for interferon induction

inhibitors of interferon are present, one which operates at a genetic level the repressor and controls the synthesis of interferon m-RNA and the other a post-transcriptional control prot[r]

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Cell-free synthesis of simian virus 40 T-antigens.

Cell-free synthesis of simian virus 40 T-antigens.

In addition to the data presented here, strong evidence in support of the model for the location of sequences coding for the SV40 T-Ags has recently come from experiments which have show[r]

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Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids.

Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids.

Results presented in this communication show that VSV proteins synthesized in vitro concurrently with genomic RNA assembled into structures that i banded in CsCl gradients or eluted from[r]

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Cell-free synthesis of functional phospholipase A1 from Serratia sp.

Cell-free synthesis of functional phospholipase A1 from Serratia sp.

moved to an expression vector containing a strong pro- moter. For example, when cloned in the pQE70 plasmid under the T5 promoter, less than 30 µg of purified PLA1 was obtained from a 100 mL culture. Although not dis- cussed in the previous reports, this appears to be due to the toxicity of the enzyme to E. coli. Because the T7 promoter used in this study is stronger than the T5 pro- moter [17–19], we expected that the induction of PLA1 expression would further decrease the yield of PLA1. Indeed, during the cultivation of E. coli transformed with the plasmid pET21a Serr PLA1, we found significant growth inhibition after induction with IPTG. A decrease in the growth rate of approximately 25 % was observed in induced E. coli as compared to a control culture without IPTG induction (Fig. 1a). Although time-course analysis of PLA1 activity indicated accumulation of functional PLA1 after IPTG induction (Fig. 1b), the expression level was too low to be confirmed by SDS-PAGE and immu- noblot analysis (data not shown). We presumed that the onset of PLA1 expression from the strong T7 promoter caused significant damage to the cells and decreased cel- lular production of proteins. This presumption was con- firmed by measuring the amounts of total cellular protein in the E. coli with or without IPTG induction. As shown in Fig.  1c, compared to non-induced E. coli, the IPTG- induced cells had approximately 25  % less total cellular protein.
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Inhibition of HeLa Cell Protein Synthesis by the Vaccinia Virion

Inhibition of HeLa Cell Protein Synthesis by the Vaccinia Virion

The very rapid inhibition of HeLa cell protein synthesis after vaccinia infection, the multiplicity effect, the ability to establish the block in the presence of actinomycin D and cycloh[r]

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