Digestion Of RNA Research Articles

Nuclease digestion of DNA and RNA in nuclei from young adult and senescent Caenorhabditis elegans (Nematoda)

Nuclei prepared from young adult and senescent Caenorhabditis elegans (Nematoda) were subjected to digestion by micrococcal nuclease and DNaseI. The kinetics of digestion of nuclei by micrococcal nuclease showed no change with age. There was, however, an age-related increase of acid-soluble deoxyribonucleotides release by DNaseI, suggesting that subtle alterations of chromatin conformation occur in aged nematodes. The ratio of nuclear RNA to DNA decreased and the nuclear RNA became more susceptible to enzymatic degradation as the worms grew old. These findings appear to indicate that nuclear RNA is less protected by protein in old nematodes. The decline of the nuclear RNA/DNA ratio with age is in good agreement with the generally accepted idea that there is a reduced level of RNA and protein synthesis in old animals.

Polyadenylic acid sequences in the genomic RNA of the togavirus of simian hemorrhagic fever

Since serologic studies have failed to relate the togavirus simian hemorrhagic fever (SHF) virus to any currently accepted genus within the Togaviridae family, the presence of polyadenylic acid (poly(A)] in the genomic RNA was analyzed in view of the different content reported for the two major genera of that family: alphaviruses where poly(A) is 40 to 120 nucleotides long and flavivirus where poly(A) is considered to be absent. Oligo(dT)-cellulose chromatography of whole genomic RNA from purified SHF virus revealed that about 36% of the molecules contained segments of poly(A) of sufficient length to bind to oligo(dT)-cellulose. However, a reproducible fraction of the RNAs did not bind to oligo(dT)-cellulose, indicating little or no poly(A) present. When analyzed by electrophoresis under denaturing conditions, both the binding and nonbinding molecules were similar in size. In addition, no polyuridylic acid [poly(U)] was detected in SHF virus genomic RNA. After digestion of the genomic RNA with pancreatic and T 1 ribonucleases, the resultant resistant polynucleotide sedimented by ultracentrifugation between tRNA and 5 S RNA. Base composition analysis of these polynucleotides detected only adenosinic residues. A mean length of 76 ∼2 nucleotides for these poly(A) sequences of SHF virus RNA was established by electrophoresis under denaturing conditions. Thus, together with previous morphological as well as biochemical findings, the presence of a poly(A) sequence is further evidence that SHF virus has distinctive characteristics which differentiates it from the two major subgroups of togavirus.

Identification of a protein covalently linked to the 5′ terminus of tobacco vein mottling virus RNA

The nature of the 5′ terminus of tobacco vein mottling virus (TVMV) RNA, a member of the potyvirus group, was investigated. Digestion of viral RNA with ribonuclease led to the appearance of a new polypeptide band of apparent molecular weight 24,000 (24 kDa) after electrophoresis on denaturing polyacrylamide gels. Viral RNA was subjected to radioiodination with the protein-specific Bolton-Hunter reagent. Radioactivity cosedimented on sucrose gradients with intact TVMV RNA. Treatment of the 125I-labeled product with the proteinase Pronase resulted in substantial loss of trichloroacetic acid-precipitable radioactivity. A radioactive species of 24 kDa was released when the 125I-labeled product was subjected to ribonuclease digestion. To localize the point of attachment, the 125I-labeled TVMV RNA was partially hydrolyzed and used as a hybridization probe against four previously described recombinant plasmids containing TVMV cDNA and a fifth plasmid containing additional 5′-terminal sequences. Hybridization was strongest to plasmids containing the most 5′-terminal sequences. Antisera against the five known proteins encoded by TVMV RNA failed to precipitate significant amounts of the new protein. This genome-linked viral protein (VPg) thus constitutes the sixth polypeptide to be associated with TVMV. It also has the largest apparent molecular weight of any RNA-linked VPg reported to date.

Neplanocin A. A cyclopentenyl analog of adenosine with specificity for inhibiting RNA methylation.

The mechanism of action of the adenosine analog, neplanocin A (NPC), was investigated in human colon carcinoma cell line HT-29. Cell viability was reduced to 38 and 17% of control by 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. Cytocidal activity was not affected by inhibition of adenosine deaminase with 2'-deoxycoformycin. Concomitant with decreased cell viability was the reduced incorporation of [14C]dThd and [3H]Leu, and to a lesser extent [3H]Urd, into acid-precipitable material. Labeling of rRNA and tRNA during drug treatment for 24 h with [methyl-3H]Met and [14C]Urd revealed that NPC primarily inhibited RNA methylation, and to a lesser extent, RNA synthesis. RNase T2 digests of total RNA indicated that base and 2'-O-methylation were inhibited to approximately the same degree. Metabolites of NPC were measured by reverse-phase high-performance liquid chromatography and it was found that the major drug metabolite was the drug analog of S-adenosylmethionine with little formation of the respective, S-adenosylhomocysteine metabolite. NPC was utilized to a very small degree for RNA synthesis where only 2 and 30 pmol of NPC/A260 were incorporated into rRNA and tRNA after 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. These results indicate that NPC is metabolized to a metabolite of S-adenosylmethionine which is a poor methyl donor for RNA methyltransferases, and that the accompanying decrease in RNA methylation and protein synthesis appears to be related to its cytocidal activity.

Specificity of initiation of plus-strand DNA by Rous sarcoma virus.

We previously reported that in the endogenous reaction of Rous sarcoma virus disrupted by melittin, plus-strand DNA initiates on a small oligonucleotide primer and that this initiation can be reconstructed in vitro in reactions containing purified minus-strand DNA as template, viral RNA as a source of primer, and reverse transcriptase (Smith et al., J. Virol. 49:200-204, 1984). Further studies on the specificity of initiation in the endogenous reaction have shown the following. (i) The primer was 12 nucleotides in length. Its sequence began with a 5' pyrimidine, followed by 11 purines, ending with rGrA-3'. This sequence was in agreement with the known plus-strand RNA sequence immediately upstream from the initiation site. Thus, the primer began one nucleotide 5' to the so-called polypurine tract that has been found on all retrovirus genomes. (ii) The transition point between RNA primer and DNA product was precisely located. It was before the end of the polypurine tract. Thus the polypurine tract, although essential for virus replication and probably a flag for the priming event, did not define the limits of the RNA primer. After primer removal, the DNA had a 5' phosphate, consistent with generation by the viral RNase H activity. The priming specificity in reconstructed reactions was also examined further, with the following observations. (i) When the source of RNA primer was prehybridized to the template viral DNA, the generation, utilization, and subsequent removal of primer were essentially the same as those observed in the endogenous reaction. In the absence of deliberate prehybridization, some specificity was lost. There were than additional locations for the 5' end of the primer as well as the transition point between RNA primer and DNA. (ii) Purine-rich oligoribonucleotides created by RNase A digestion of viral RNA could prime strong-stop plus DNA, but again with the loss of specificity relative to that in the endogenous reaction. (iii) The 5' end of the minus-strand DNA template was not required for initiation of strong-stop plus DNA. Therefore, the specificity of initiation did not depend upon an intramolecular interaction requiring the two inverted repeat sequences that flank the long terminal repeat.

Vesicular stomatitis virus mutant with altered polyadenylic acid polymerase activity in vitro.

In vitro RNA synthesis by purified virions of a stock of tsG16(I) was aberrant compared with that of wild-type (wt) vesicular stomatitis virus. RNA made in vitro by tsG16(I) contained a larger proportion of A residues in polyadenylic acid [poly(A)] tracts than did RNA synthesized by wt virus, tsG13(I), tsG21(II) or tsG41(IV). Experiments to determine whether the aberrant polyadenylation was correlated with the known thermolability of the tsG16(I) L protein were inconclusive. Total product RNA made by tsG16(I) was methylated to almost the same extent as wt RNA, contained the same major methylated 5' cap structure as wt RNA, and was translated as well in a reticulocyte cell-free system, yielding the same molecular weight proteins in similar ratios. Most polyadenylated [poly(A)+] RNA made by tsG16(I) was considerably larger than wt poly(A)+ RNA and richer in AMP:UMP residues; however, the protein-coding capacities of mutant and wt poly(A)+ RNAs were similar. This suggested that most mRNAs made in vitro by tsG16(I) might possess very long poly(A)+ tracts, and digestion of RNA by T1 RNase supported this. It appeared, therefore, that a virally coded component of vesicular stomatitis virus could affect polyadenylation. This could be the poly(A) polymerase itself, a protein involved in control of polyadenylation, or a protein which affects an event spatially and temporally connected with polyadenylation (such as initiation of the subsequent mRNA).

Polyadenylylated nuclear RNA contains branches.

A highly charged component can be isolated from a total RNase T2 digest of nuclear polyadenylylated RNA from HeLa cells that is separable from caps by (dihydroxyboryl)aminoethyl-cellulose chromatography. Chemical and enzymatic analyses show that the component contains a 2'-5' phosphodiester bond that creates a branch at the 2'-hydroxyl group of one nucleotide already linked to an adjoining nucleotide through the usual 3'-5' phosphodiester bond. [Formula: see text] This structure was confirmed by analysis of a similar component isolated from nuclease P1 digests of the same nuclear polyadenylylated RNA. Branches occur in roughly 10% of nuclear polyadenylylated RNAs, including those >10S in size, but are absent from cytoplasmic polyadenylylated RNA. Possible implications for branches as intermediates in mRNA processing are discussed.

Use of micrococcal nuclease in the purification of highly template dependent RNA-dependent RNA polymerase from brome mosaic virus-infected barley

The template dependence of RNA-dependent RNA polymerase from brome mosaic virus (BMV)-infected barley was greatly increased by micrococcal nuclease digestion of the endogenous RNA. [ 32P]UMP incorporation by the nuclease-treated enzyme was stimulated 20-fold when BMV RNA was added as template, while incorporation by the untreated enzyme was stimulated only 5-fold by the addition of BMV RNA. Other properties of BMV polymerase were not changed significantly by nuclease digestion. The extract remained highly active and template specific. Analysis of the products of the reaction showed that separated BMV RNA components could be replicated independently to yield full-length replicative-form RNAs. These data provide strong evidence that the extract is capable of initiating RNA synthesis and that it includes the intact viral replicase. This method should be of general use, allowing the study of cell-free replication of any viral nucleic acid without requiring purification or solubilization of the replicase.

Different RNA patterns of globin and non-globin 40S heterogeneous nuclear RNA-protein complexes in chicken reticulocyte nuclei.

40s heterogeneous nuclear RNA-protein complex (HnRNP) was isolated from chicken reticulocyte nuclei after digestion of RNA by the endogenous nuclease. The size of the total RNA and beta globin RNA was compared by agarose and high resolution acrylamide gel electrophoresis. The results show that size of the total RNA ranges between 20 to 650 bases and the most prominent sizes are between 20 and 70-80 bases. The size of the RNA increases at roughly 10 base intervals between 40 and 70 bases on an acrylamide gel. The size of the beta globin RNA is smaller (25-50 bases) than the total RNA and also the RNA pattern is considerably different from that of total RNA. This suggests that, although the overall structure of 40s HnRNP might be the same, there is significant differences in the interaction of globin RNA with HnRNP protein and that of non-globin RNA with HnRNP protein in chicken reticulocyte nuclei.

A Covalently Linked Protein at the 5'-Ends of the Genomic RNAs of Pea Enation Mosaic Virus

SUMMARY Evidence ispresented for the presence of a covalently linked protein that isattached to the 5'-ends of genomic RNA of pea enation mosaicvirus ( PEMV). Nuclease digestion of PEMV RNA released protein with anapparent mol. wt. of approx. 17 500. The absence of a 5'-capand the inability to introduce [32P]phosphate into the5'-termini of these RNAs suggests that the protein iscovalently bound to PEMV RNA.

Molecular structure of a new family of ribonucleases.

Bacillus amyloliquefaciens produces a ribonuclease1 (barnase), the function of which is probably the digestion of external RNA: it is excreted by the bacillus and within the cell its action is inhibited by a protein of ∼89 residues to which it binds with high affinity2. Determination3 of the amino acid sequence of the ribonuclease, which is a monomer of molecular weight 12,382 consisting of 110 residues, has revealed its homology with other prokaryotic and eukaryotic ribonucleases4–6. We have now determined the atomic structure of barnase by X-ray crystallographic analysis. We report that its structure includes a large central β-pleated sheet and two α-helices. The arrangement of these secondary structures is different from that found in bovine pancreatic ribonuclease.

Does tobacco mosaic virus RNA contain cytokinins?

Tobacco mosaic virus was previously reported to contain between 5 and 17 modified nucleosides with cytokinin activity per RNA molecule. Using a new and highly sensitive method for gas chromatography of permethylated cytokinins, we were unable to detect cytokinins in enzymic or alkaline digests of TMV RNA. The method was sensitive enough to assay 1 cytokinin nucleoside per 10 TMV RNA molecules. Soybean callus bioassay also failed to detect activity in TMV RNA hydrolysates which corresponded to any of the cytokinin nucleosides known to occur in RNA. We therefore suggest that TMV RNA does not contain cytokinin nucleosides.

Interactions of Qβ replicase with Qβ RNA

The interactions of Qβ replicase with Qβ RNA were investigated by treating replicase-Qβ RNA complexes under various conditions with ribonuclease T 1, and by characterizing enzyme-bound RNA fragments recovered by a filter binding technique. Evidence for replicase binding at two internal regions of Qβ RNA was obtained. One region (at about 1250 to 1350 nucleotides from the 5′ end) overlaps with the initiation site for coat protein synthesis; this interaction is thought to be inessential for template activity but rather to be involved in the regulation of protein synthesis. Binding to this site (called the S-site) requires moderate concentrations of salt but no magnesium ions. The other region (at about 2550 to 2870 nucleotides from the 5′ end) is probably essential for template activity; binding to this site (called the M-site) is dependent on the presence of magnesium ions. The nucleotide sequences of the RNA fragments from the two sites were determined and found to have no common features. Under the conditions tested, replicase binding at the 3′ end of Qβ RNA could not be demonstrated, except when initiation of RNA synthesis was allowed to occur in the presence of GTP and host factor. If instead of intact Qβ RNA, a complete RNAase T 1 digest of Qβ RNA was allowed to bind to replicase, oligonucleotides from the S-site and the M-site, and oligonucleotides from a region close to the 3′ end, were found to have the highest affinity to the enzyme. The RNA fragments recovered in highest yield, M-2 and S-3 from the M and S-site, respectively, were isolated on a preparative scale and their enzyme binding properties were studied. In competition assays with random RNA fragments of the same size, selective binding was observed both for the M and the S-site fragment. Partial competition for replicase binding was found if M-2 and S-3 were presented simultaneously to the enzyme. Either fragment, if preincubated with replicase, caused a specific inhibition of initiation of Qβ RNA-directed RNA synthesis, without inhibiting the poly(rC)-directed reaction. The results are discussed in terms of a model of replicase-Qβ RNA recognition. Template specificity is attributed to binding of internal RNA regions to replicase, resulting in a specific spatial orientation of the RNA by which the inherently weak, but essential, interaction at the 3′ end is allowed to occur and to lead to the initiation of RNA synthesis.

Ribonuclease of Entamoeba invadens

Two bands of RNAase activity were observed on gel electrophoresis of fractions obtained during partial purification of the RNAase of Entamoeba invadens. The faster migrating band which was responsible for most of the RNAase activity was isolated by gel electrophoresis. A p I of 5.5 was determined for this RNAase activity and estimates of its molecular weight and sedimentation coefficient gave values of about 25 000 and approximately 2.8 S. It exhibited maximum activity around pH 6, and digestion of RNA showed a pattern expected of an endoribonuclease.

Fusion of Semliki forest virus with red cell membranes

The interaction of Semliki Forest virus (SFV) and red cells was studied using biochemical and immunological methods. When [ 3H]uridine-labeled SFV was adsorbed at 0°, pH 5.8, to ribonuclease-loaded human red cell ghosts, the viral RNA became sensitive to ribonuclease within 2 to 5 min at 20–37°, but not at 0°. Maximal digestion of RNA was achieved within 30 min at 37° suggesting that the nucleocapsids had penetrated the red cell membranes. When the red cells that had been fused with [ 35S]methionine-labeled SFV were treated with trypsin, about 70% of E2 protein and 50% of E1 protein was digested, demonstrating that the viral envelope proteins remained on the surface of the red cell. This was also shown by radioimmunoassay, by complement-dependent immune hemolysis assay, and by the ability of the red cells to bind Staphylococcus aureus bacteria to the cell surface after treatment with anti-envelope antiserum. When the red cells carrying SFV glycoproteins on their surfaces were transferred to pH 5.8, the cells were readily fused after the temperature had been raised to 42°. This shows that the fusion of virus envelope with red cell membranes precedes the fusion of red cells.

Ethidium bromide: a nucleic acid stain for tissue section.

The phenanthridinium dye, ethidium bromide (EB), selectively intercalates into double-stranded regions of nucleic acids with a large and specific increase in fluorescence. When used for the staining of fixed tissue sections, the dye stains cellular nuclei with excellent resolution of microscopic detail. In some fixed tissues, particularly pancreatic acini, cytoplasm stains intensely and this staining can be abolished by digestion with trypsin and ribonuclease. The orange fluorescence of EB can be easily distinguished from the green fluorescence of fluorescein and EB is thus an excellent counterstain for immunofluorescence. Ethidium bromide is a useful and practical stain for the fluorescence microscopy of tissue sections and, in combination with enzymatic digestion of RNA, provides a simple way to differentially localize DNA and RNA.

Structural analysis of templates and RNA polymerase III transcripts of Alu family sequences interspersed among the human β-like globin genes

Cloned DNA fragments from the human β-like globin genomic region can be transcribed in vitro by RNA polymerase III. We have investigated the structure of two templates and their transcripts by DNA sequencing, size fractionation of ribonuclease T1 generated oligonucleotides, and ribonuclease H digestion of RNA: DNA duplexes. The data indicate that repetitive DNA sequences, members of the Alu family of interspersed 300 bp reiterated DNA, are imbedded in both templates. The RNAs transcribed from them are composed of an entire Alu family sequence at their 5′ ends linked to 3′ ends of non-repetitive sequence.

Nucleotide sequence of 5S ribosomal RNA from the posterior silk glands of Bombyx mori.

A low molecular weight RNA fraction was obtained from the posterior silk glands of Bombyx mori. 5S rRNA was purified from this fraction by gel filtration on a column of Sephadex G-100 and electrophoresis on a discontinuous polyacrylamide gel devised for large scale preparation. The nucleotide sequence of this RNA was determined mainly by a chemical sequencing method on polyacrylamide gels using RNA labeled with [32P]pCp at the 3' terminus (1). An enzymatic sequencing method on gels using 32P-labeled RNA at the 5' end (2) and conventional sequence analyses of the complete digests of the unlabeled RNA with RNase T1 [EC 3.1.27.3] and RNase A [EC 3.1.27.5] were used for complete determination of the total sequence. 16 base substitutions were observed between B. mori and Drosophila melanogaster 5S rRNAs.

Identification of small polyhedral virus particles in thin sections of plant cells by an enzyme cytochemical technique

An enzyme cytochemical method for distinguishing particles of small polyhedral plant viruses containing single-stranded RNA from those of ribosomes in thin sections of infected cells is described. The method was completely satisfactory for plant cells infected by six of the nine viruses tested. Particles of each of these retained their encapasidated RNA and hence its densely staining properties, when aldehyde-fixed tissues were treated with RNase under conditions appropriate for the digestion of RNA from cytoplasmic ribosomes. However, in cells infected by three of the viruses studied, all of which are known to synthesize empty protein shells, enzyme treatment also removed some of the virus-encapsidated RNA. It is concluded that although the method used has considerable merit for cytopathological studies of cells infected by viruses, it should be used with caution.

Preferential binding of 3'-terminal fragments of alfalfa mosaic virus RNA 4 to virions.

3'-Terminal fragments present in a partial ribonuclease T1 digest of RNA 4 of alfalfa mosaic virus were selectively bound to virions of the same virus by incubation in 10 mM sodium phosphate and 1 mM EDTA, pH 7.0. The virions with the associated fragments were separated from the remainder of the digest by velocity gradient centrifugation. When the incubation was performed in 27.7 mM Na2HPO4 and 6.2 mM citric acid, pH 6.8, plus 10% glycerol, much more, but less specific, binding was obtained. However, in this case the terminal fragments present in unbound material of the digest were almost exclusively 5' termini. The four RNA species species of alfalfa mosaic virus have an extensive 3'-terminal homology. Therefore, it is expected that specific virion binding can be used to obtain 3'-terminal fragments of different lengths of the three genome RNA species (RNAs 1, 2, and 3) of alfalfa mosaic virus and, possibly, of related viruses. Such fragments are of great importance for the study of the specific 3'-terminal interaction with coat protein subunits which is thought to be responsible for the activation of the viral genome.