Yeast viral double-stranded RNAs have heterogeneous 3′ termini

The yeast virus, ScV, is communicated only by mating. It has two separately encapsidated dsRNAs. One of these, L, codes for the major capsid polypeptide. The other, M, codes for a polypeptide toxic to yeasts without ScV-M particles. Defective interfering particles containing fragments of M (S) displace ScV-M when they arise. We have shown that five independently isolated S dsRNAs are all derived by internal deletion of M. The 3′ ends of all the ScV dsRNAs are markedly heterogeneous. For instance, half of the first 35 nucleotides at one 3′ end of M and S are variable. Conserved sequences at the 3′ ends of M and S are AAACACCCAUCA_OH and AUUUCUUUAUUUUUCA_OH. Conserved sequences at the 3′ ends of L are UAAAAAUUUUUCA_OH and AAAAAUXCA_OH, where X is variable. We propose that the sequence AUUUUUCA_OH is a recognition sequence for the capsid-associated single-stranded RNA polymerase activity. Since all the viral RNAs have pppGp 5′ termini, their 3′ termini probably extend one nucleotide beyond the terminal pppGp.     

Methylation status of 13S ribosomal RNA from hamster mitochondria: the presence of a novel riboside, N4-methylcytidine.

The ribosomal RNA ("13S" RNA) of the small ribosomal subunit of hamster cell mitochondria has been found to have a distinctive pattern of methylated residues. Each molecule contained, on the average, approximately one residue of m4Cp, m5Cp and m5Up, and two residues of m62Ap. The natural occurrence of m4Cp has not previously been reported; we propose that this nucleotide is homologous to its ribose-methylated congener, m4Cmp, which is characteristic of bacterial 16S ribosomal RNA. We detected neither m4Cp nor m4Cmp in the hamster cell cytoplasmic ribosomal RNA. This is the first documentation of a modified residue present in mitochondrial RNA but absent from the cytoplasmic RNA of the same cells.     

Micro thin-layer techniques for rapid sequence analysis of P-labeled RNA: Double digestion and pancreatic ribonuclease analyses

Double digestion of oligonucleotides obtained from ribonuclease T₁ or pancreatic ribonuclease A fingerprints results in the following series of products: (Ap)_0-nCp, (Ap)_0-nUp, and (Ap)_0-nGp. A new micromethod is described for the rapid analysis of these digests. The procedure consists of two-dimensional chromatography on a small PEI thin-layer plate. In the first dimension, the oligonucleotides are separated independent of their Ap content into three groups, which represent the Cp-, Gp-, and Up- 3′-terminal oligonucleotide series, respectively. In the second dimension, the products are fractionated according to their chain length. This method, which allows direct identification of even the longer Ap tracts in a double digest of an oligonucleotide or an RNA chain, is very rapid and highly sensitive and can be applied to the simultaneous analysis of a large number of samples in a single run. The procedure has also been adapted to the analysis of pancreatic ribonuclease A digests of small RNA fragments.     

Chemical and genetic analysis of 16S ribosomal RNA in Escherichia coli

Summary Comparative chemical analyses of oligonucleotides arising from pancreatic RNase digestions of 16S ribosomal RNAs from Escherichia coli strains K12 and B(H) showed that a decanucleotide fragment, (5Ap,4Gp)Cp, could be detected exclusively in strain K12 but not in strain B(H), in spite of gross similarity of nucleotide distributions between the two strains.The K12-specific oligonucleotide could not be cotransduced with streptomycin and/or spectinomycin resistant markers from K12 to B(H) by phage Plkc, indicating that the genes specifying 16S ribosomal RNA are not closely linked to these markers on the chromosome.     

Comparative Study of the Ribosomal RNA from Leishmania and Trypanosoma1

The ribosomal RNA from several stocks of the genera Leishmania and Trypanosoma were studied by gel electrophoresis, sedimentation on sucrose density gradients and RNA/DNA hybridization experiments. Three major components were observed after electrophoresis in polyacrylamide gels (PAGE-SDS), the relative molecular masses being respectively: X₁= 0.83 megadaltons, X₂= 0.63 megadaltons and X₃= 0.54 megadaltons for Leishmania RNA; and X₁= 0.86 megaldaltons, X₂= 0.78 megadaltons, and X₃= 0.58 megadaltons for Trypanosoma RNA. Depending upon the isolation procedure, a fourth component. X₀= 1.2 megadaltons (26S), became evident. The later component was purified from Leishmania brasiliensis (Y) by centrifugation on a linear 15-30% sucrose density gradient. This component, after heat denaturation and PAGE-SDS, gave rise to two bands coinciding in molecular mass with those of X₂ and X₃ indicating that these components are part of the large ribosomal subunit whereas X₁ belongs to the small one. The above mentioned differences in mobilities of components X₁ and X₂ between the two genera were no longer observed after electrophoresis in denaturing agarose-formaldehyde gels, suggesting secondary structural differences among these RNA species. Hybridization experiments with L. brasiliensis (Y) DNA showed that both RNA types compete equally well for the ribosomal sites in this DNA, and that L. brasiliensis (Y) rRNA recognizes the ribosomal sites in DNA of Trypanosoma cruzi (EP), thus indicating that no gross changes occurred in their nucleotide sequences during evolution.     

Purification and properties of bacteriophage T4-induced RNA ligase

RNA ligase has been extensively purified by a new procedure in high yield from T4-infected Escherichia coli. The enzyme consists of a single polypeptide chain of molecular weight 47,000. It catalyzes the formation of a phosphodiester bond between a 5′-PO₄-terminated oligonucleotide and a 3′-OH terminated oligonucleotide. The purified enzyme catalyzes both the intramolecular formation of single-stranded circles with longer oligonucleotides of the type pAp(Ap)_nA_?OH, where n is about 15 or greater and the intermolecular joining of pAp(Ap)₃A_OH (where the 5′-PO₄-terminated oligonucleotide is short enough to prevent apposition of its 3′ and 5′ ends) to UpUpU_OH when high concentrations of the 3′-OH-terminated acceptor oligonucleotide are present. Preparations of RNA ligase at all stages of purification show an unusual dependence of specific activity of the enzyme on the concentration of enzyme present in the assay. However, when care is taken to determine meaningful specific activities at each step, the ligase is found to be very stable during chromatography on various ion-exchange columns and may be purified by conventional techniques.     

Polyethylene Oxide (PEO) and Polyethylene Glycol (PEG) Polymer Sieving Matrix for RNA Capillary Electrophoresis

The selection of sieving polymer for RNA fragments separation by capillary electrophoresis is imperative. We investigated the separation of RNA fragments ranged from 100 to 10,000 nt in polyethylene glycol (PEG) and polyethylene oxide (PEO) solutions with different molecular weight and different concentration. We found that the separation performance of the small RNA fragments (<1000 nt) was improved with the increase of polymer concentration, whereas the separation performance for the large ones (>4000 nt) deteriorated in PEG/PEO solutions when the concentration was above 1.0%/0.6%, respectively. By double logarithmic plot of mobility and RNA fragment size, we revealed three migration regimes for RNA in PEG (300-500k) and PEO (4,000k). Moreover, we calculated the smallest resolvable nucleotide length (N_min) from the resolution length analysis.     

Structural features unique to the 5 S ribosomal RNAs of the thermophilic cyanobacterium Synechococcus lividus III and the green plant chloroplasts

The complete nucleotide sequence of the 5 S ribosomal RNA from the thermophilic cyanobacterium Synechococcus lividus III was determined. The sequence is: ^5′U-C- C-U-G-G-U-G-G-U-G-A-U-G-G-C-G-A-U-G-U-G-G-A-C-C-C-A-C-A-C-U-C-A-U-C- C-A-U-C-C-C-G-A-A-C-U-G-A-G-U-G-G-U-G-A-A-A-C-G-C-A-U-U-U-G-C-G-G-C- G-A-C-G-A-U-A-G-U-U-G-G-A-G-G-G-U-A-G-C-C-U-C-C-U-G-U-C-A-A-A-A-U-A- G-C-U-A-A-C-C-G-C-C-A-G-G-G-U_OH^3′This 5 S RNA has regional structural characteristics that are found in the green plant chloroplast 5 S RNAs and not in other known sequences of 5 S ribosomal RNAs. These homologies suggest a close phylogenetic relationship between S. lividus and the green plant chloroplasts.     

The low molecular weight of RNAs of adenovirus 2-infected cells

The cytoplasm of HeLa cells infected with adenovirus type 2 contains many species of low molecular weight RNA, including several of viral origin. In addition to a 9 S messenger RNA, the viral genome gives rise to two species of virus-associated RNA: the major species is 5.5 S RNA or virus-associated RNA_I, and the minor species is 5.2 S RNA or virus-associated RNA_II. Virus-associated RNA_I occurs in the cytoplasm in several electrophoretically separable forms, and its sequences are also present in high molecular weight nuclear RNA but not in cytoplasmic mRNA. The structure of virus-associated RNA_II is shown to be distinct from that of the major species, and the position of its gene is mapped on the viral genome. The two virus-associated RNA genes are located on the r strand near position 30 of the adenovirus type 2 physical map, and are separated by a spacer of about 75 base-pairs.     

Nucleotide sequence of nuclear 5S RNA of mouse cells

The nucleotide sequence of nuclear 5S RNA of mouse cells was determined. The 5S RNA is 117 nucleotides long with one mole each of m₃^2,2,7G, Gm, Am and Cm, two moles of Um, and three moles of ψ as modified nucleosides, and it is rich in uridylate residues (about 36 %). The 5′-terminal hexanucleotide-containing cap structure, m₃^2,2,7GpppAm-Um-A-C-U-, is identical with that of U1 RNA. This RNA contains sequences complementary to the terminal sequences of the introns of heterogeneous nuclear RNAs.     

Characterization of the ribosome-protected regions of 125I-labelled rabbit globin messenger RNA

The fragments of ¹²⁵I-labelled rabbit globin messenger RNA protected from pancreatic RNAase by initiating 40 S subunits and 80 S ribosomes were analysed using the techniques of RNA sequencing. The fragments were cleaved specifically at cytidine residues generating oligonucleotides labelled in their 3′ terminal residue. Analysis of the partial digestion products of these oligonucleotides after treatment with pancreatic, T₁, U₂ and T₂ RNAase enabled their sequences to be deduced. Sequences were determined from knowledge of the specificities of the ribonucleases and then confirmed in a separate analysis making use of the known electrophoretic mobilities of each base. This combination of methods served to establish that the 40 S- and 80 S-protected fragments are related, and that both contain the initiation codon of the mRNA. The 80 S-protected fragment is about 40 bases in length whilst the 40 S-protected fragments range from 50 to more than 60 bases in length. The most prominent of these 40 S-protected fragments is about 50 bases in length and extends more towards the 5′ end of the mRNA than does the 80 S-protected fragment. It follows that 80 S ribosomes do not protect the 5′ end of the mRNA from nuclease digestion and that the 5′ terminus of rabbit globin mRNA must be at least 15 to 30 bases from the initiation codon.     

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25.

An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonuclease-resistant part of the native 5S RNA of Escherichia coli, at 0 degrees C. A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 25 degrees. Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0 degrees C and 25 degrees C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions. Protein L18 initially protected most of the 5S RNA against ribonuclease digestion, at 0 degrees C, but was then gradually released prior to the formation of the larger RNA fragment. It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18.     

Isolation and analysis of (Gp)nXp sequences of rat liver 5S RNA by means of restricted ribonuclease T2 hydrolysis

Essentual difficulties arise when base number in oligoguanylic blocks and location of these blocks along the polynucleotide chain need to be determined in the course of determination of the nucleotide sequences in ribonucleic acids. To overcome this difficulty it is suggested to take advantage of a recently discovered resistance of phosphodiester bond between kethoxalated G and its 3′-neighbour against T₂ RNase hydrolysis 1,2. The approach is illustrated by analysis of 5S RNA from rat liver. Sequences of general formula (Gp)_nXp were isolated from T₂ RNase hydrolysate of 5 S RNA rapidly and quantitatively. The information obtained greatly facilitates the whole procedure of sequencing. It is expected that the method proposed would be effective for analysis of 5 S and 4 S RNA and for highmolecular weight fragments of ribosomal and viral RNAs.     

Equimolar addition of oligoribonucleotides with T4 RNA ligase.

T4 induced RNA ligase will join equimolar concentrations of two oligoribonucleotides, (Ap)3C and p(Up) 5, to form a single product, (Ap)3Cp(Up) 5, in high yield. The presence of the 3' phosphate on p(Up)5 prevents the oligomer from adding to itself. The pH optimum of the reaction is about 7.5, but less of the undesirable adenylated intermediate, App(Up) 5, forms at pH 8.2. The reaction rate is a linear function of oligomer concentration from 3 micronM to 0.6 mM. The data suggest that T4 RNA ligase will be a useful enzyme for the synthesis of oligomers of defined sequence.     

Sequence complexity of poly(A) RNA fromPhysarum polycephalum

Summary Poly(A) RNA from S phase, G₂ phase and starved macroplasmodia of Physarum contain mRNA sequences which when translated in vitro, yield similar patterns of polypeptides after fluorography.Reassociation of nick-translated DNA (Cot) allows the isolation of highly labeled single copy DNA which, after saturation hybridization with poly(A) RNA, gives values of 23% for growth and 17% for starvation.Homologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that 22–28% of the genome is transcribed during growth and 12% during starvation and that about half of the cDNA reacts with 0.1% of the genome and could represent 50–80 RNA species, each present in about 1,000 copies per nucleus. Up to 25,000 different RNA species, 1–5 copies each per nucleus, are estimated to be present during growth, and about 15,000 during starvation. Heterologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that the RNA sequences in S and G₂ phase of the cell cycle are similar, with RNA sequences being more abundant in G₂ phase.During starvation about 25% of the sequences present during growth cannot be detected and those sequences present during growth have become diluted during starvation.     

The effect of growth temperatures on the in vivo ribose methylation of Bacillus stearothermophilus transfer RNA

When Bacillus stearothermophilus was cultured at 70 and at 50 °C, 1.4 times as many methyl groups were incorporated into tRNA produced at the higher temperature compared to that produced at the lower. This was due predominantly to a threefold increase in the 2′-O-methylribose moieties of the tRNA. The type and quantity of the base methylated nucleotides in the tRNAs produced in cultures grown at 70 and 50 °C were almost identical. The base methylated nucleotides found were: m²Ap, ms²Ap, ms²i⁶Ap, an unidentified i⁶Ap derivative, m⁶Ap, m₂⁶Ap, m¹Gp, m⁷Gp, m⁵Up-(Tp), and an unidentified methylated Up or Cp.³ The nucleotide m⁷Ap, never before reported to be a constituent of tRNA, has been tentatively identified as a component of B. stearothermophilus tRNA.     

The determination of the species classification of Baikal planarian cocoons found in the stomach of the black grayling (Thymallus arcticus baicalensis) by a comparative analysis of the nucleotide sequences of the ribosomal RNA gene]

Comparative analysis of nucleotide sequences of gene 18S of ribosome RNA was carried out. The results show that the genetic sequences of the given locus could be used as a molecular marker to identify the species of planaria irrespective of ontogenetic stage. The articles deals with problem of specific determination of cocoons of Baikal planaria from the stomach of Baikal black grayling using comparative analysis of nucleotide sequences of ribosome RNA fragments with known sequences determined earlier for Baikal planaria. The cocoons belong to two species of Rimacephalus. The authors discuss also the importance of feeding relationships of planaria and benthophage fish to investigate the biotic factors that influence the evolution of Baikal planaria.