Showing papers on "RNA-dependent RNA polymerase published in 1982"
TL;DR: It is proposed that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions.
2,077 citations
TL;DR: Duck hepatitis B virus, a DNA virus closely related to human hepatitis Birus, was studied in infected duck liver and a pathway for the replication of the DNA genome of hepatitis B-like viruses by reverse transcription of an RNA intermediate is proposed.
1,374 citations
TL;DR: Four RNA transcripts encoded by cauliflower mosaic virus DNA have been detected in the polyadenylated RNA from virus-infected turnip leaves, two of which have the same 5' termini, at nucleotide 7435.
347 citations
TL;DR: SP6 RNA polymerase possesses a stringent promoter specificity similar to, but distinct from that of the other phage RNA polymerases, and is also highly active in synthesis of poly(rG) with poly(dI) .
262 citations
TL;DR: The results confirmed that the most abundant cytoplasmic RNA species in such cells is specified by a small region of the genome defined by the EcoRI J fragment.
Abstract: Cytoplasmic RNA prepared from five lymphoid cell lines and a Burkitt lymphoma biopsy was radioactively labeled in vitro and hybridized to cloned EcoRI restriction endonuclease fragments of B95-8 Epstein-Barr virus DNA. The results confirmed that the most abundant cytoplasmic RNA species in such cells is specified by a small region of the genome defined by the EcoRI J fragment. Detailed mapping experiments precisely localized these transcripts within the sequence of the rightmost one-third of the EcoRI J fragment. DNA sequencing suggested that this region of the Epstein-Barr virus genome is unable to code for protein. The major early transcripts consisted of two non-polyadenylated RNA species, each about 170 nucleotides in length. They were both transcribed off the same strand of the DNA and showed significant sequence homology with each other. The coding sequences of the two small RNAs contained potential intragenic control regions for RNA polymerase III.
195 citations
TL;DR: It is concluded that the availability of the beta-gamma bond of ATP is an indispensable requirement for faithful and specific in vitro initiation only by RNA polymerase II in the whole cell extract.
181 citations
TL;DR: The transcriptional elongation reaction carried out by calf thymus RNA polymerase II in vitro differs in several respects from that which must take place in vivo.
174 citations
TL;DR: In vitro elongated viral RNA appeared with a peak of 5S in sucrose gradients and hybridized preferentially to a promoter-proximal region of SV40 DNA, and this RNA was found to map between the major initiation site at nucleotide 243 and nucleotides 335-337.
173 citations
TL;DR: To investigate the time of onset of transcriptional activity in the early mammalian embryo, incubated one-cell pronuclear embryos with 3H-adenosine and labelled three labelled RNA species, finding no label was found in ribosomal RNA at the one- cell stage but synthesis of mature rRNA species was evident in the two-cell embryo.
Abstract: The time of onset of transcriptional activity in the early mammalian embryo is unclear. RNA synthesis has been shown to occur at the two-cell stage in the mouse1–4 and at the two- to four-cell stage in the rabbit5, but the period immediately following fertilization has proved largely inaccessible to biochemical characterization of transcription because of the low permeability of early embryos to exogenous precursors such as 3H-uridine1–7. This difficulty, and the failure to detect RNA polymerase activity in the pronuclei of one-cell mouse embryos8, suggested that the embryonic genome was transcriptionally inactive until some time after the first cleavage. To investigate this issue further, we have now incubated one-cell pronuclear embryos with 3H-adenosine, which is taken up about 1,000 times faster than uridine3. Three labelled RNA species could be identified. The major product is large, heterodisperse, poly(A)− RNA. High-molecular-weight poly(A)+ RNA is also heavily labelled but this is mostly due to cytoplasmic polyadenylation of previously non-polyadenylated, stored RNA. A significant amount of label is incorporated into the -CCA termini of transfer RNA but some new synthesis of tRNA also takes place. No label was found in ribosomal RNA at the one-cell stage but synthesis of mature rRNA species was evident in the two-cell embryo.
150 citations
TL;DR: The results are consistent with the conclusion that the observed sequential transcription of vesicular stomatitis virus mRNAs is due to obligatory entrance of all polymerases at the leader gene, and suggest that the transcriptase and replicase may recognize the same promoter.
149 citations
TL;DR: Heterologous hybridisations between the cloned mouse 7S sequence and RNAs prepared from rat, human and chick cells have shown that the non-B1 part of the 7S RNA molecule has been highly conserved during recent eucaryotic evolution.
Abstract: A cDNA library has been prepared from mouse embryo small RNAs and screened for the presence of clones complementary to the highly abundant cytoplasmic 7S RNA. One clone (pA6) was selected which hybridized exclusively with 7S RNA on a Northern blot prepared from cytoplasmic RNA run on high resolution polyacrylamide/urea gels. Sequence analysis of this clone has shown that at least 65 nucleotides at the 5' end of 7S RNA are extensively homologous with the highly repeated mouse B1 family. Heterologous hybridisations between the cloned mouse 7S sequence and RNAs prepared from rat, human and chick cells have shown that the non-B1 part of the 7S RNA molecule has been highly conserved during recent eucaryotic evolution. There are multiple copies of 7S RNA genes in the genomes of mouse, human, rat and chick cells, but substantial differences exist in copy number and genomic organisation in these organisms.
TL;DR: It is suggested that Ro 09-0179 interferes with some process of viral replication which occurs between viral uncoating and the initiation of viral RNA synthesis.
Abstract: Ro 09-0179 (4',5-dihydroxy-3,3',7-trimethoxyflavone), isolated from a Chinese medicinal herb, was found to have potent antiviral activity. It selectively inhibited the replication of human picornaviruses, such as rhinoviruses and coxsackieviruses in tissue culture, but not other DNA and RNA viruses. Ro 09-0298 (4',5-diacetyloxy-3,3',7-trimethoxyflavone), an orally active derivative of Ro 09-0179, prevented coxsackievirus (B1) infection in mice. The critical time for the inhibition of rhinovirus replication by Ro 09-0179 was 2 to 4 h after virus adsorption, i.e., in the early stages of virus replication. It markedly inhibited coxsackievirus and rhinovirus RNA synthesis in infected HeLa cells, but not in a cell-free system using the RNA polymerase complex isolated from the infected cells. In the infected cells, the RNA polymerase complex was not formed in the presence of Ro 09-0179. Therefore, it is suggested that Ro 09-0179 interferes with some process of viral replication which occurs between viral uncoating and the initiation of viral RNA synthesis.
TL;DR: In this article, the authors characterized the viral-specific RNAs present in C127 mouse cells transformed by the BPV-1 virus or by cloned DNA and found that all five viral RNA species share a common 3′ terminus at 0.34 map units and the 5′ ends map at approximately 0.85 map units.
TL;DR: It is shown that bacteriophage T4 has two alternative mechanisms to initiate DNA replication; one dependent on Escherichia coli RNA polymerase (RNA nucleotidyltransferase, EC 2.7.6), and onedependent on general recombination.
Abstract: We show that bacteriophage T4 has two alternative mechanisms to initiate DNA replication; one dependent on Escherichia coli RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6), and one dependent on general recombination. Continued DNA synthesis under recombination-defective conditions was sensitive to rifampin, an inhibitor of RNA polymerase. On the other hand, DNA synthesis accelerated in spite of the present of rifampin if recombination occurred.
TL;DR: The gene encoding the RNA subunit (M1 RNA) of RNAase P (EC 3.26.5) from Escherichia coli has been isolated, and its complete nucleotide sequence, including flanking regions, has been determined.
TL;DR: It is found that the S4 double-stranded RNA segment (the segment that encodes the σ3 polypeptide, the major outer capsid polypeptic) is responsible for the capacity of type 2 to inhibit L cell RNA and protein synthesis.
TL;DR: The observation that most RNA synthesis parameters are temperature invariant despite the increase ofppGpp suggests that the mechanism of RNA synthesis control by ppGpp, assumed to involve an interaction of RNA polymerase wtihpp, is itself temperature dependent such that, with increasing temperature, higher concentrations of pp Gpp are required to affect the RNA polymerases.
Abstract: For Escherichia coli B/r growing in glucose minimal medium, the following parameters of RNA synthesis remained invariant between 20 and 40 degrees C: RNA polymerase concentration (RNA polymerase/mass), rRNA and tRNA concentration (RNA/mass), RNA polymerase activity (fraction of total RNA polymerase actively engaged in RNA chain elongation), and stable RNA synthesis relative to total RNA synthesis. The following parameters increased 3.4-fold over the same temperature range: rRNA chain elongation rate, guanosine tetraphosphate (ppGpp) concentration, and culture growth rate. Above 40 degrees C, the changes became more complex, and the growth rate began to decrease. The observation that most RNA synthesis parameters are temperature invariant despite the increase of ppGpp suggests that the mechanism of RNA synthesis control by ppGpp, assumed to involve an interaction of RNA polymerase wtih ppGpp, is itself temperature dependent such that, with increasing temperature, higher concentrations of ppGpp are required to affect the RNA polymerase.
TL;DR: The results illustrate the structural evolution of eukaryotic RNA polymerases at the subunit level and the immunological relationships of form B RNA polymerase from yeast, plant, insect, crustacea, and mammal.
TL;DR: These results further support the concept that the nuclear matrix may function in the localization and the structural organization of (viral) heterogeneous nuclear RNA during its processing.
TL;DR: All of the mouse hepatitis virus-specific RNAs are transcribed from a single species of negative-stranded RNA template of genomic size, suggesting that all of the virus- specific mRNAs could be released from this RNA species upon heating.
Abstract: There are seven virus-specific mRNA species in mouse hepatitis virus-infected cells (Lai et al., J. Virol. 39:823-834, 1981). In this study, we examined virus-specific negative-stranded RNA to determine whether there are corresponding multiple negative-stranded RNAs. Intracellular RNA from mouse hepatitis virus-infected cells was separated by agarose gel electrophoresis, transferred to nitrocellulose membranes, and hybridized to positive-stranded genomic 60S [32P]RNA. Only a single RNA species of genomic size was detected under these conditions. This RNA was negative stranded. No negative-stranded subgenomic RNA was detected. We also studied double-stranded replicative-form RNA in the infected cells. Only one replicative-form of genomic size was detected. When the double-stranded RNA isolated without RNase treatment was analyzed, again only one RNA species of genomic size was detectable. Furthermore, most of the virus-specific mRNAs could be released from this RNA species upon heating. These results suggest that all of the mouse hepatitis virus-specific RNAs are transcribed from a single species of negative-stranded RNA template of genomic size.
TL;DR: Protein C is shown here to be cleaved by two different mechanisms, which were distinguished by their sensitivity to dilution, and it is proposed that the dilution-sensitive cleavage of protein C is due to a virus-coded protease, probably p22 itself, and that the Dilution-independent cleavage isdue to intramolecular self-cleavage ofprotein C.
Abstract: It has previously been shown that when encephalomyocarditis viral RNA is translated in cell-free extracts of rabbit reticulocytes, it synthesizes a virus-coded protease, p22, which is derived by cleavage of a precursor protein, C. Protein C is shown here to be cleaved by two different mechanisms, which were distinguished by their sensitivity to dilution. One mechanism was sensitive to dilution; the other was not. The biphasic cleavage behavior was unchanged by diluting incubation mixtures with untranslated reticulocyte extract instead of buffer, suggesting that both types of cleavage were mediated by virus translation products. It is proposed that the dilution-sensitive cleavage of protein C is due to a virus-coded protease, probably p22 itself, and that the dilution-independent cleavage is due to intramolecular self-cleavage of protein C.
TL;DR: RNA-dependent RNA polymerase activity was found in mouse hepatitis virus strain A59 (MHV-A59)-infected cells and it was probable that the enzymes represent two different species ofRNA polymerase and perform different roles in virus-specific RNA synthesis.
Abstract: RNA-dependent RNA polymerase activity was found in mouse hepatitis virus strain A59 (MHV-A59)-infected cells. The enzyme was induced in the infected cells and could not be detected in the MHV-A59 virion. Two peaks of RNA polymerase activity, one early and the other late in infection, were detected. These polymerase activities were in temporal sequence with early and late virus-specific RNA synthesis. Both of them were found to be associated with membrane fractions. There were significant differences in the enzymatic properties of the two polymerases. The early polymerase, but not the late polymerase, could be activated by potassium ions in the absence of magnesium ions and also had a lower optimum pH than the late polymerase. It was therefore probable that the enzymes represent two different species of RNA polymerase and perform different roles in virus-specific RNA synthesis. The effects of cycloheximide on MHV-specific RNA synthesis were determined. Continuous protein synthesis was required for both early and late RNA synthesis and might also be required for shutoff of early RNA synthesis.
TL;DR: The results strongly suggest that the 2.3-kilobase RNA molecule is the mRNA of the major polypeptide of the envelope, which carries the viral surface antigen determinants.
Abstract: Mouse L cells transformed with recombinant plasmids carrying hepatitis B virus (HBV) DNA fragments were used to study the transcription of the viral surface antigen gene (gene S). An HBV-specific, polyadenylated, 2.3-kilobase RNA was mapped on the HBV genome. This RNA hybridized with approximately 75% of the genome and excluded the region of the HBV core antigen gene (gene C). The 2.3-kilobase RNA species was present only in cell lines that produced hepatitis B surface antigen. An HBV-specific 2.3-kilobase RNA was also detected in human hepatoma cell line PLC/PRF/5 which produced hepatitis B surface antigen. A study of gene S expression in the transformed mouse L cells allowed us to localize the regions of initiation and termination of gene S transcription. Our results strongly suggest that the 2.3-kilobase RNA molecule is the mRNA of the major polypeptide of the envelope, which carries the viral surface antigen determinants.
TL;DR: The protein homology is sufficient, however, for deduction of the correct coding phase of the RNA and allows the alignment of the corresponding nucleic acid sequence data from different alphaviruses without knowledge of the sequence of the entire genomes.
Abstract: The alphaviruses produce two mRNAs after infection: the genomic (49S) RNA which is translated into the nonstructural (replicase) proteins and the subgenomic (26S) RNA which serves as the mRNA for the virion structural proteins. The sequence of the region of the genomic RNA that contains the 5' end of the subgenomic RNA and the 5' flanking sequences in the genomic RNA were determined for several alphaviruses. A highly conserved sequence of 21 nucleotides was found which includes the first two nucleotides of the subgenomic RNA and the 19 nucleotides preceding it. We propose that the complement of this sequence in the minus strand is the recognition site used by the viral transcriptase for initiation of transcription of 26S RNA and that, in general, such short recognition sequences are commonly used among the RNA viruses. The COOH-terminal sequence of the nonstructural polyprotein precursor has been deduced for each virus. These protein sequences are highly homologous and are followed by multiple in-phase termination codons clustered in the nontranslated region of the 26S RNA in each case. In contrast to the proposed transcriptase recognition site, the particular triplets used for a given conserved amino acid have diverged markedly during evolution of these viruses. The protein homology is sufficient, however, for deduction of the correct coding phase of the RNA and allows the alignment of the corresponding nucleic acid sequence data from different alphaviruses without knowledge of the sequence of the entire genomes.
TL;DR: The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murines hepatitis virion RNA have been investigated by cell-free translation and it has been shown that RNA 7 codes for the nucleocapsid protein, RNA 6 code for the E1 protein, and RNA 3 codes forThe E2 protein.
Abstract: The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These studies have shown that RNA 7 codes for the nucleocapsid protein, RNA 6 codes for the E1 protein, RNA 3 codes for the E2 protein, and RNA 2 codes for a 35,000-dalton nonstructural protein. Genomic RNA directs the cell-free synthesis of three structurally related polypeptides of greater than 200,000 in molecular weight.
TL;DR: Compared the 3'-terminal non-coding sequences of the RNAs from 10 alphaviruses are compared and this region is found to be composed of distinct domains in terms of base composition, degree of sequence conservation, and sequence organization.
TL;DR: The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods the authors have used to determine size).
Abstract: The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).
TL;DR: The complete sequence of the small (S) viral RNA species of snowshoe hare (SSH) bunyavirus has been determined, principally from a DNA copy of the RNA cloned in the E.coli plasmid pBr322.
Abstract: The complete sequence of the small (S) viral RNA species of snowshoe hare (SSH) bunyavirus has been determined, principally from a DNA copy of the RNA cloned in the E.coli plasmid pBr322. The viral S RNA (negative sense strand) is 982 nucleotides long (3.3 x 10(5) daltons) with complementary 5' and 3' end sequences. It has a base composition of 30.5%U, 25.8%A, 24.9%C and 18.7%G. In the viral complementary (plus sense) strand there are two overlapping open reading frames initiated by methionine codons. One reading frame codes for a 26.8 x 10(3) dalton protein, the other for a 10.5 x 10(3) dalton protein. The larger gene product is presumably related to the viral nucleoprotein (N) that is coded by the S RNA (Gentsch and Bishop (1978) J. Virol. 28, 417-419). The smaller gene product is probably related to the recently identified S RNA coded nonstructural protein (NSS) induced in virus infected cells (Fuller and Bishop (1982) J. Virol. 41, 643-648).