In Vitro Continuation of RNA Synthesis Initiated In Vivo

Nucleic Acids ◽  
2003 ◽  
pp. 161-168
Author(s):  
Theodore Gurney
Keyword(s):  
Rna Synthesis

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

Synthesis of acid-soluble spore proteins by Bacillus subtilis

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss
Keyword(s):  
Bacillus Subtilis ◽  
Maximum Rate ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Spore Proteins ◽  
Major Acid

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

Cellular heterogeneity of uridine incorporation in collecting tubules: effect of DOCA

1985 ◽  
Vol 248 (4) ◽  
pp. F552-F564
Author(s):  
A. Vandewalle ◽  
F. Cluzeaud ◽  
M. Chavance ◽  
J. P. Bonvalet
Keyword(s):  
Rna Synthesis ◽  
Cellular Heterogeneity ◽  
Nuclear Surface ◽  
Intercalated Cells ◽  
Uridine Incorporation ◽  
Collecting Tubules ◽  
Silver Grains ◽  
Stimulation Of

In previous studies we showed that in vitro uridine incorporation along the renal tubule is heterogeneous and that DOCA induces a stimulation of RNA synthesis in distal cortical and medullary structures. The present work examines by autoradiography of isolated tubules and renal tissue sections the cellular heterogeneity of the connecting (CNT) and cortical collecting (CCT) tubules after in vivo injection of [3H]uridine in normal and DOCA-treated rabbits. Data confirmed the profile of uridine incorporation along the tubule, which was found in in vitro experiments, and the DOCA-induced stimulation of RNA synthesis. In microdissected CNT and CCT of control kidneys, statistical analysis of the distribution of labeling revealed the presence of two distinct cell populations: one with low labeling (2-3 silver grains per nucleus) and one with high labeling (10-13), which represent 64 and 36%, respectively (CNT), and 74 and 26%, respectively (CCT), of the whole population. Histological data showed that the respective proportions of intercalated cells (29% in CNT; 21% in CCT) and connecting tubule cells (65%) or principal cells (79%) are close to those of the populations with high or low labeling. In addition, autoradiographs on renal sections directly demonstrated that the labeling of intercalated cells (19.3 silver grains/100 micron2 nuclear surface in CNT; 14.7 in CCT) was three times higher than that of connecting (6.6) or principal (5.8) cells. In isolated CNT and CCT, DOCA induced similar absolute increases in the labeling of the two populations. However, the relative increase was more than two times higher in the population with low labeling (+131% in CNT, +210% in CCT) than in the one with high labeling (+71% and +98%). We conclude that cell population of the collecting cortical tubule (CNT and CCT) is heterogeneous with regard to uridine incorporation, reflecting RNA synthesis.

Ribonucleic acid synthesis inPlasmodium knowlesimaintained bothin vivoandin vitro

Parasitology ◽  
1975 ◽  
Vol 71 (2) ◽  
pp. 199-209 ◽  
Author(s):  
P. I. Trigg ◽  
P. G. Shakespeare ◽  
Susan J. Burt ◽  
Sally I. Kyd
Keyword(s):  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Nuclear Division ◽  
Plasmodium Knowlesi ◽  
Acid Synthesis ◽  
Agarose Gel Electrophoresis ◽  
Trophozoite Stage ◽  
Late Trophozoite

RNA extracted from purified parasites ofPlasmodium knowlesiwas fractionated using agarose gel electrophoresis. Preparations from parasites grown bothin vivoandin vitrocontained species of RNA with sedimentation coefficients of 4·0S, 5·0S, 16·6S, 24·2S, 31·4S, 38·0S and 48·3S. There was less RNA present in parasites grownin vitrothan the equivalent stage parasites grownin vivobut the proportional amounts of the various species of RNA was similar in both cases. It is suggested that the 24·2S and 16·6S species of RNA are ribosomal and that the high molecular weight 31·4S, 38·0S and 48·0S species are ribosomal precursors. Ribosomal RNA synthesis occurs throughout the cell cycle during growth from the ring to the schizont stage; maximum incorporation of [H3]-adenosine occurs at the late trophozoite stage before nuclear division.

scholarly journals Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

2017 ◽  
Vol 115 (2) ◽  
pp. E162-E171 ◽  
Author(s):  
François Ferron ◽  
Lorenzo Subissi ◽  
Ana Theresa Silveira De Morais ◽  
Nhung Thi Tuyet Le ◽  
Marion Sevajol ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Mutation Rates ◽  
Bifunctional Enzyme ◽  
Antiviral Compound ◽  
Mismatch Correction ◽  
Limited Efficacy

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium

2010 ◽  
Vol 88 (2) ◽  
pp. 529-539 ◽  
Author(s):  
Simon Stammen ◽  
Franziska Schuller ◽  
Sylvia Dietrich ◽  
Martin Gamer ◽  
Rebekka Biedendieck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacillus Megaterium ◽  
Protein Production ◽  
Rna Synthesis ◽  
Escherichia Coli Phage

scholarly journals Significance in Replication of the Terminal Nucleotides of the Flavivirus Genome

2003 ◽  
Vol 77 (19) ◽  
pp. 10623-10629 ◽  
Author(s):  
Alexander A Khromykh ◽  
Natasha Kondratieva ◽  
Jean-Yves Sgro ◽  
Ann Palmenberg ◽  
Edwin G Westaway
Keyword(s):  
Rna Synthesis ◽  
Point Mutations ◽  
In Vitro Activity ◽  
Rna Dependent Rna Polymerase ◽  
Stem Loop ◽  
Double Stranded Rna ◽  
Positive Strand ◽  
Proposed Model

ABSTRACT Point mutations that resulted in a substitution of the conserved 3′-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3′-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3′-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of φ6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3′-penultimate cytidines and a 3′-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3′-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

scholarly journals Mechanism of Stimulation of Plus-Strand Synthesis by an RNA Replication Enhancer in a Tombusvirus

2005 ◽  
Vol 79 (15) ◽  
pp. 9777-9785 ◽  
Author(s):  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Dual Function ◽  
Long Distance ◽  
Replication Assay ◽  
Rna Interaction ◽  
Stimulation Of

ABSTRACT Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.

scholarly journals SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

2004 ◽  
Vol 78 (23) ◽  
pp. 13153-13162 ◽  
Author(s):  
Keum S. Choi ◽  
Akihiro Mizutani ◽  
Michael M. C. Lai
Keyword(s):  
Mammalian Cells ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Affinity Purification ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

ACTION OF KI, THYROXINE AND CYCLIC AMP ON [3H]URIDINE INCORPORATION INTO THE RNA OF THYROID SLICES

1976 ◽  
Vol 83 (2) ◽  
pp. 313-320 ◽  
Author(s):  
Mario A. Pisarev ◽  
Leonardo O. Aiello ◽  
Diana L. Kleiman de Pisarev
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Action ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Protein Biosynthesis ◽  
Rna Degradation ◽  
Precursor Pool ◽  
Rna Precursor

ABSTRACT Potassium iodide (KI) has been shown to impair thyroid protein biosynthesis both in vivo and in vitro. The present study was performed in order to clarify its mechanism of action. Ribonucleic acid (RNA) synthesis was studied in beef thyroid slices with either [32P] or [3H]-uridine as labelled precursors. Both KI and thyroxine (T4) at 10−5 m significantly decreased RNA labelling under our conditions. In other experiments RNA degradation was examined in pulse-labelled and actinomycin D-treated slices. KI did not modify the degradation of the [3H]-RNA thus indicating that it interferes with the biosynthesis rather than with the degradation of RNA. Taking the perchloric acid soluble radioactivity as a rough index of the precursor pool the present results would indicate an action at this level. Both KClO4 and methylmercapto-imidazole relieved the gland from the inhibitory action of KI, supporting the view that an intracellular and organified form of iodine is responsible for this action. Since T4 also reproduced the effects of KI on RNA synthesis we would like to propose iodothyronines as the intermediates of this action. Cyclic AMP has been shown to stimulate thyroid protein biosynthesis. The present results demonstrate an action at the RNA level. Cyclic AMP increased both the PCA-soluble and RNA-linked radioactivity, thus suggesting an effect at the RNA precursor pool. KI at 10−5 m blocked the action of 2 mm cyclic AMP.

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