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PubMed1975

Transcription of the genome of adenovirus type 12. I. Viral mRNA in abortively infected and transformed cells.

In baby hamster kidney (BKH-21) cells abortively infected with adenovirus type 12, polysome-associated, virus-specific RNA could be detected starting 5 to 7 h after infection. The amount of this RNA reached a maximum between 10 to 12 h after infection and continued to be synthesized at a reduced level until late in infection (48 to 50 h.). In BHK-21 cells transformed by adenovirus type 12 (HB cells), 0.26% of the polysome-associated mRNA was virus specific. The size of the virus-specific mRNA isolated from polysomes of BHK-21 cells abortively infected with, or transformed by adenovirus type 12 was determined by electrophoresis in polyacrylamide gels in 98% formamide, i.e., under conditions which eliminated secondary structure or aggregation of RNA. In abortively infected hamster cells viral mRNA size classes of molecular weights 0.9 times 10-6 and 0.65 times 10-6 to 0.67 times 10-6 were predominant. A minor fraction of 1.5 times 10-6 daltons was consistently found and increased with time after infection. Late after infection (24 to 26 h), viral mRNA of 1.9 times 10-6 daltons was also observed. The size distribution of adenovirus type 12-specific mRNA from transformed hamster cells (HB line) was very similar to that in abortively infected cells, except that the relative amount of the viral mRNA fraction of 1.5 times 10-6 daltons was much higher. It is uncertain whether the viral mRNA of high-molecular-weight represents mixed transcripts derived from integrated viral genomes and adjacent host genes.

Adenoviridae↗
PubMed1979

Structural states of dictyostelium myosin.

Myosin purified from Dictyostelium amoebae has approximately 10% by weight of RNA associated with it, unless specific steps (DEAE cellulose chromatography or RNase digestion) are taken to remove it. This RNA has significant effects on the structural states formed by the myosin at low ionic strength in the presence of Mg2+. Rapid precipitation of RNA-free myosin by dilution generates bipolar thick filaments (540 nm long, 33 nm thick), often with a bare zone and a 15-nm transverse repeat. Rapid precipitation of myosin with copurified RNA yields linear aggregates of bipolar filaments, showing some lateral association. Slow precipitation of RNA-free myosin by dialysis yields very long filaments or ribbons (greater than 5 micrometer, 30--60 nm wide) in which the myosin may be packed diagonally across the filament, similar to the "side-polar" aggregates formed by other nonmuscle myosins and by smooth muscle myosin (Craig R, Megerman J: J Cell Biol 75:990, 1977; Hinssen H, D'Haese J, Small JV, Sobieszek A: J Ultrastruct Res 64:282, 1978). Slow precipitation of myosin with copurified RNA generates linear filaments with repeat intervals of 290 and 650 nm. Other polyanions were tested for their effects on myosin aggregation. Total RNA and ribosomal RNA from Dictyostelium, when added to RNA-free myosin, also induced the extensive linear aggregation seen with the copurified RNA/myosin complex, although higher concentrations of RNA were required to obtain quantitatively the same effect. DNA and heparin were also effective inducers of linear aggregation, whereas homopolymers of nucleotides and of acidic or basic amino acids were poorly effective.

Dictyostelium↗
PubMed1979

Kinetics and mechanism of tobacco mosaic virus assembly: direct measurement of relative rates of incorporation of 4S and 20S protein.

The mechanism of assembly of tobacco mosaic virus has been investigated under conditions in which the rates of incorporation of the 4S and 20S proteins can each be directly measured by analytical centfrifugation. Under these conditions, pH 6.5, 6.5 degrees C, 0.10 M ionic strength potassium orthophosphate, the protein can be made to exist as a metastable 20S aggregate that is necessary for efficient reconstitution. The overall assembly process consists of an initiation (nucleation) reaction that requires two to three 20S disk aggregates per RNA molecule and is followed by an elongation (growth) reaction. In the elongation phase of assembly the 4S protein is incorporated 50 to 70 times faster than the 20S disk, calculated on the basis of a steady-state kinetic analysis. Therefore, under these conditions, in which the rate of assembly is about 0.06 of that at pH 7, 20 degrees C, 0.10 M ionic strength orthophosphate, the 4S protein preferentially participates in the elongation phase. At this slow reconstitution rate intermediate assembly states (about 70-168 S) can be observed. The kinetics of both protein incorporation and nucleoprotein formation suggest that the elongation process is composed of at least two different, possibly sequential, rate-limiting reactions.

Kinetics↗
PubMed1978

Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.

Arenaviridae↗
PubMed1977

Comparison of hydrophobic and strongly hydrophilic cleavable crosslinking reagents in intermolecular bond formation in aggregates of proteins or protein-RNA.

Most of the bifunctional reagents in protein chemistry possess a strongly hydrophobic backbone, derived from aliphatic or aromatic hydrocarbons. Even bifunctionals of more than 30 A in length of this sort form intramolecular bridges preferentially. In recent years, the intermolecular crosslinking of physiological protein aggregates has gained in importance. As shown in the crosslinking of hemoglobin with two sets of hydrophobic and strongly hydrophilic reagents, derived from azo dyes and tartaric acid, respectively, in this case it is not primarily the length of the bifunctional, but the hydrophilic structure that will enhance intermolecular crosslinking. Artificial dimers of native structure may be obtained. For the crosslinking of RNA to protein, we have synthesized a new reagent, 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid p-nitrophenyl ester. In a two step reaction, it is attached to adenine and cytosine moieties at pH 6 first, and to lysine side chains at pH 7,5. The reagent has been applied to the poly-A sequence of globin messenger RNA nucleoprotein.

Adenosine↗
PubMed1976

Defective interfering passages of Sindbis virus: nature of the defective virion RNA.

Defective interfering particles of Sindbis virus contain 20S RNA identical to that found in BHK cells co-infected with standard and defective virions. We have characterized these RNAs by their oligonucleotide fingerprints. Most of the oligonucleotides were identical to those found in the mRNA (26S RNA) that codes for the virion structural proteins. Three oligonucleotides found in 20S RNA were absent from the 26S RNA pattern and may represent sequences from the 5' end of the virion RNA. Previous difficulties in describing the nature of the defective virion RNA were due to the aggregated state of the RNA. Nucleocapsids obtained from standard and defective virions were essentially the same size and had about the same density, suggesting that defective particles contain more than a single molecule of 20S RNA.

Base Sequence↗
PubMed1975

Sequence of a specifically encapsidated RNA fragment originating from the tobacco-mosaic-virus coat-protein cistron.

When 25-S tobacco mosaic virus (TMV) protein aggregate and TMV RNA, which has been partially digested by T1 RNase, are mixed under conditions suitable for reconstitution, only a few RNA fragments are encapsidated. These fragments were isolated and purified by polyacrylamide gel electrophoresis. The sequence of the three main fragments, the longest of which (fragment 1) was estimated to contain 103 nucleotides, has been determined. The two smaller fragments are portions of the longer chain produced by an additional specific scission. Because of the great affinity of 25-S TMV protein for this nucleotide sequence, it will be referred to as the "specifically encapsidated RNA fragment". The occurrence of a "hidden break" in the sequence has been demonstrated: fragment 1, purified by electrophoresis on a polyacrylamide gel without 8 M urea, gives rise upon further electroporesis in the presence of urea to two new bands corresponding to the two halves of the molecule. A stable hair-pin secondary structure has been derived from the base sequence which can account for the specificity of action of the enzyme. Because of its properties, we have suggested elsewhere that the sequence of fragment 1 might correspond to the disk recognition site for reconstitution, which is known to be located at the 5' end of the intact RNA. But experiments with TMV RNA whose 5'-OH end has been radioactively phosphorylated with polynucleotide kinase show that this is not the case. Analysis of the amino acid coding capacity of the fragment has instead revealed that fragment 1 is a portion of the TMV coat protein cistron.

Base Sequence↗
PubMed1975

Characterization of putative nuclear pre-messenger RNA of Ehrlich ascites tumor cells.

Ehrlich ascites tumor cell putative nuclear pre-messenger RNA (pre-mRNA) was isolated under conditions minimizing RNA degradation by ribonucleases, aggregation, and non-specific protein-RNA interaction. Isolated under these conditions, it sedimented 10 to 12 S; proteinase K, a powerful proteolytic enzyme with a broad action spectrum, gave similar sedimentation values and polyacrylamide gel electrophoresis revealed the major component migrating ahead of 16S E. Coli rRNA marker. Cesium chloride buoyant density analysis of pre-mRNA revealed 2 components (1.51 and 1.68 g/cm3). Therefore, pre-mRNA appeared to be smaller than some previous reports.

Animals↗
PubMed1978

Kinetics of net RNA degradation during development in Dictyostelium discoideum.

Based on dry weight, first-order kinetics adequately describe the net decrease of RNA in Dictyostelium discoideum NC-4 until culmination, when a cessation of RNA degradation occurs. Between aggregation and sorocarp formation, the cells lose 40% of their RNA, or 19 mM-glucose equivalents expressed in terms of cell volume at aggregation. In sorocarps the spores contain a disproportionately large amount of RNA. Net RNA contents in sorocarps and in spores are unaffected by the availability of exogenous glucose. The relationship of dry weight, cell number and packed cell volume during differentiation is given.

Dictyostelium↗
PubMed1978

Effect of estrogen on gene expression in the chick oviduct. In vitro transcription of the ovalbumin gene.

Problems involved in using the Hg-nucleotide technique for in vitro chromatin transcription are 2-fold. First, Escherichia coli RNA polymerase can utilize endogenous RNA as template and synthesize complementary sequences which remain base-paired to the template, thereby allowing it to bind to the SH-Sepharose column and copurify with the newly synthesized Hg-RNA. Second, non-mercurated endogenous RNA can bind to the SH-Sepharose through aggregation with Hg-RNA and thus be retained in the final RNA preparation. These two problems associated with the Hg-nucleotide technique can be minimized by modifying the conditions for RNA synthesis and SH-Sepharose chromatography. Using the modified procedure the Hg-nucleotide and SH-Sepharose technique can remove more than 90% of endogenous RNA contaminants. In order to directly demonstrate that the mRNAov sequences detected in vitro result from de novo transcription of oviduct chromatin, experiments were carried out which show that the hybridizable RNA sequences contain the Hg element and that the synthesis of these RNA sequences is sensitive to low concentrations of actinomycin D. These combined results strongly suggest that the majority of mRNAov sequences detected by hybridization to cDNAov is indeed due to DNA-dependent RNA synthesis by E. coli RNA polymerase and not due to an artifact of endogenous RNA contamination. This observation was further supported by data obtained using a filter hybridization method which measures directly the mRNAov sequences present in [3H]RNA synthesized from chromatin. The 3H-labeled ovalbumin messenger RNA was assayed by hybridization to cloned pOV230 DNA containing the ovalbumin structural gene sequence. With this modified Hg-nucleotide-SH-Sepharose technique and filter hybridization technique, we have restudied the in vitro transcription of the ovalbumin gene from chromatins isolated at different stages of hormone-induced oviduct development. The results are in agreement with our previous findings which suggest that the primary regulation of ovalbumin synthesis by steroid hormones occurs at the transcriptional level.

Animals↗
PubMed1978

Biochemical and electron microscopic characterization of DNA-RNA complexes from HeLa cell mitochondria.

The previous electron microscopic investigations on the occurrence in HeLa cell mitochondria of transcription complexes of mitochondrial DNA [Aloni, Y., and Attardi, G. (1972a), J. Mol. Biol. 70, 363-373] have been extended with the aim of obtaining these complexes in a reasonably pure form for biochemical analysis. By using conditions designed to minimize losses of such structures and any possible contamination by nuclear DNA, it has been shown that a substantial fraction (40 to 50%) of mitochondrial DNA can be isolated from exponentially growing HeLa cells in the form of fastsedimenting complexes with RNA. These complexes have been characterized with respect to density and sedimentation properties, content in newly synthesized RNA, stability of the association of RNA with DNA, presence of different forms of mitochondrial DNA, and electron microscopic appearance. The properties of these complexes, as well as the results of reconstruction experiments, strongly suggest that the majority of such structures represent true transcriptional intermediates. The occurrence in this fraction of replicating or newly replicated mitochondrial DNA molecules has been observed. Although the presence of single-stranded DNA segments makes the replicative intermediates particularly susceptible to aggregation with free RNA, electron microscopic observations point to the possibility that these intermediates may be recruited for transcription.

DNA, Mitochondrial↗
PubMed1976

Assembly of a spherical plant virus.

The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 degrees C and did not require the presence of Mg2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.

Capsid↗
PubMed1976

The region of tobacco mosaic virus RNA involved in the nucleation of assembly.

The interaction of TMV RNA with the disk aggregate of TMV protein at the initiation of assembly has been studied by using the techniques of RNA sequencing. The 5' end group has been identified, and shown not to be protected in the early stages of assembly from accessibility to nuclease digestion. A population of RNA fragments of average length 250 nucleotides, originating from a unique region of TMV RNA, is encapsidated by limited assembly, and sufficient sequence information is available to identify certain unusual features. The protected region does not contain highly reiterated simple repeating sequences, but may contain more complicated repeats. The length and complexity of the nucleation region may reflect adaptation to the efficient mediation of the conformational change from disk to helix of TMV protein, besides a requirement for binding to the disk, and this may be an important part of the mechanism of specificity in the nucleation of assembly.

Electrophoresis↗
PubMed1977

Assembly of tobacco mosaic virus in vitro: elongation of partially reconstituted RNA.

It was reported that the rate of elongation of partially reconstituted RNA, if prepared without centrifugation, showed dependency upon the state of protein aggregation like that found when free RNA was used as the nucleic acid source [Butler, P. J. G. (1974) J. Mol. Biol. 82, 333-341]. Butler concluded from this result that disks are the kinetically favored protein source for the elongation of the tobacco mosaic virus rod. We show that partially reconstituted RNA prepared without centrifugation always contains a significant amount of free RNA and conclude that the dependency of its elongation rate upon the state of protein aggregation is due to contamination of his partially reconstituted RNA preparation by free RNA. The elongation rate of partially reconstituted RNA, which had been pelleted and contained no free RNA, was found to be about the same regardless of the state of aggregation of the added protein.

Centrifugation, Density Gradient↗
PubMed1978

Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor.

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

Amanitins↗
PubMed2026

Riboregulation: a non-canonical tau function.

Almost since its discovery, tau protein has perplexed scientists and clinicians with its varied roles in physiology as well as its appearance as phosphorylated protein aggregates of various structures in many neurodegenerative diseases. Tau plays a role in microtubule stabilization, but from the earliest of studies, tau has also been observed to bind to RNA, with recent research suggesting tau has a higher affinity for some RNA species compared to microtubules. In the context of disease, tau dysfunction potentiates disruptions to RNA metabolism, including the perturbation of mRNA splicing, impairment of translation, de-repression of transposable elements, and alteration of RNA export and degradation. Tau aggregates directly sequester diverse RNA species and RNA binding proteins. Emerging evidence reinforces the characterization of tau as an RNA binding protein, highlighting questions about both the physiological and disease-related functions of this direct RNA binding. The disparate structure of tau in normal and various disease states makes teasing apart the various impacts on RNA and regulation a more difficult puzzle requiring future study. In this review, we summarize the evidence for tau's role in RNA biology, including as an RNA binding protein.

tau Proteins↗
PubMed1975

Murine oncornavirus high-molecular-weight RNA structure thermal stepwise dissociation of 70S murine leukemia-sarcoma virus to subunits and low-molecular-weight associated RNAs.

The thermal dissociation into subunits and low-molecular-weight (LMW) associated RNAs of the aggregate structure of 70S RNA of a murine leukemia sarcoma viral complex was studied. By polyacrylamide-agarose gel electrophoresis, it was found that at low temperature a fraction of the genome was converted into an intermediate population of RNA (Im.P) with an apparent molecular weight of 6.6 times 10-6. At higher temperature, the 70S RNA and the Im.P RNA were successively dissociated into two RNA subunits called "I" and "II" and 70S-associated LMW RNAs. The apparent molecular weight of subunit I was about 5 times 10-6 and that of subunit II was about 3.2 times 10-6. The release of 4S, 5S, 5.5S, and 8S RNAs from 70S RNA at various temperatures was studied by composite polyacrylamide gel electrophoresis. It was found that the nature of hydrogen bonding to the 70S RNA was different for each LMW RNA species. A possible relationship of the association between the subunits and each 70S-associated LMW RNA, based on their T-m values, is discussed.

Electrophoresis, Polyacrylamide Gel↗