Kin-ichiro Miura

A preliminary investigation on the molecular structure of rice dwarf virus ribonucleic acid

X-Ray diffraction and infrared absorption measurements have been made of a ribonucleic acid obtained from rice dwarf virus which was isolated from the infected rice leaves. On a fibre diagram obtained at 75% relative humidity, 52 independent reflections were observed. All of them are indexed on a hexagonal lattice with a = 40·0 A and c = 30·5 A. It was found that the unit cell contains three molecules, each of which is displaced along the c-axis direction by c /3 from the neighbours. Intensities of the reflections were used to calculate the cylindrically symmetrical Patterson function. From this Patterson function it was concluded that the ribose-phosphate chains form two intertwined helices one of which is displaced along the helix axis by 13·0 A from the other. Each helix has its axis parallel to the c -axis, a pitch of 30·5 A, and ten nucleotide residues per turn. On the basis of the infrared dichroism observed for the 1225 cm −1 and 1084 cm −1 bands, the orientation of the PO 2 − group has been determined. The O…..O line makes an angle of about 70° and the bisector of 2 − group in the A and B forms of DNA.

Segments of genome of viruses containing double-stranded ribonucleic acid☆

Abstract Double-stranded RNA preparations which were obtained from cytoplasmic polyhedrosis virus of silkworm, rice dwarf virus and reovirus have been analyzed by polyacrylamide gel electrophoresis and ultraviolet scanning. Ten segments were recognized consistently for polyhedrosis virus and reovirus, and 12 for rice dwarf virus. The presence of such genome segments seems to be a common characteristic of double-stranded viral RNA. The size distribution of the genome segments is specific to each virus. The molecular weight of a segment ranged from 0.35 to 2.6 × 10 6 for polyhedrosis virus, from 0.44 to 2.8 × 10 6 for rice dwarf virus, and from 0.63 to 2.6 × 10 6 for reovirus. The sum of the molecular weights of a set of these fragments (1.46 × 10 7 for polyhedrosis RNA, 1.53 × 10 7 for rice dwarf RNA and 1.49 × 10 7 for reovirus RNA) corresponds to the calculated maximum molecular weight of RNA in a virion. Electron microscopic observation of the size distribution of these RNA preparations confirms the results of gel electrophoresis.

Double-stranded ribonucleic acid from rice dwarf virus

Abstract Ribonucleic acid (RNA) was extracted with phenol treatment from the rice dwarf virus (RDV) isolated from infected rice plants. This RNA has a complementary base composition, that is, both the ratios of (adenine/uracil) and (guanine/cytosine) are nearly unity. It contains 44% guanine plus cytosine. It appears as threads when precipitated in alcohol. A hyper-sharp schlieren pattern was obtained in the ultracentrifuge, as seen for DNA. The ultraviolet absorption of this RNA in 0.0015 M NaCl-0.00015 M sodium citrate showed a sharp increase when the temperature was about 80°. RDV-RNA did not react with formaldehyde at 37°, but after it was heated at 100° for 10 minutes and then cooled quickly it reacted with formaldehyde. RDV-RNA was resistant to ribonuclease digestion, as compared with ribosomal RNA or transfer RNA. It is concluded that the RNA of RDV is double-stranded.

Optical rotatory dispersion and circular dichroism of rice dwarf virus ribonucleic acid

Abstract The optical rotatory dispersion and circular dichroism of a double-stranded ribonucleic acid from rice dwarf virus have been measured. The RNA exhibited multiple Cotton effects with two peaks (282 and 228 mμ) and two troughs (250 and 220 mμ) between 210 and 350 mμ, which resembled the profiles of normal RNAs. The circular dichroic spectrum showed an intense positive band at 260 mμ, close to the cross-over point of the Cotton effect. The magnitude of the Cotton effect and the intensity of the circular dichroic band are the greatest ever observed in nucleic acids. Heat denaturation drastically reduces the magnitude of the 282 mμ peak and 250 mμ trough and causes shifts to the red of their positions. The denaturation curve measured by the change in height of the 282 mμ peak gives a melting temperature of 79 °C in 1/100 standard saline citrate solution, with a sharp transition region which provides evidence of a doublestranded helical structure. The melting temperature measured using the circular dichroic band at 260 mμ was 79 °C, in good agreement with the value 81 °C from the hyperchromicity at 258 mμ.

[68] Preparation of bacterial DNA by the phenol-pH 9-RNases method

Publisher Summary This chapter describes the method for the extraction of DNA from bacterial cells using phenol. Phenol treatment is considered one of the most effective methods for deproteinization. Whereas a neutral or acidic buffer (pH 5-7) extracts RNA into aqueous phase, rather than DNA from bacterial cells, in phenol treatment slightly alkaline buffer (pH 9) extracts DNA with a little RNA contamination. The DNA is further purified by the simultaneous use of two kinds of ribonucleases, pancreatic ribonuclease I plus ribonuclease T1, to eliminate RNA contamination. The purified DNA preparation thus obtained is highly polymerized (molecular weight 1-2 X 107), and has high transforming activity.

Specificity in the Structure of Transfer RNA

Publisher Summary This chapter discusses the recent work on the specificity embodied in the structure of transfer RNA (tRNA). The chapter also discusses the base composition of tRNA. The numerous analyses of the base composition of bulk tRNA preparations from different sources have been reviewed in this chapter. The various arrangements of nucleotides in tRNA are also discussed in this chapter. Counter-current distribution, column chromatography, and other separation techniques have been used in the fractionation of tRNAs. Some of them are highly specific for one particular amino acid. The base composition of these fractionated tRNAs is characteristic. The arrangement of nucleotides in tRNA, such as (a) nucleotide distribution, (b) anticodon sequence, and (c) terminal nucleotide sequences, are discussed in this chapter. Although a definite tertiary structure of tRNA has not yet been defined, each specific tRNA, which has a specific base composition, may have a specific tertiary configuration, because the melting profiles of a few specific tRNAs differ from each other. The chapter also describes various three-dimensional structure of tRNA. The chapter concludes with the modification of nucleic acid bases, without scission of the polynucleotide chain, and the resulting change in the biological activities of nucleic acids from viruses or bacteria. In order to elucidate the functional sites of tRNA, it seems logical to examine the change in the activity of such modified tRNA.

HETEROGENEITY IN THE NUCLEOTIDE SEQUENCE NEAR THE AMINO ACID-ACCEPTING TERMINAL OF TRANSFER RNA.

A transfer RNA charged with a 14C (or 3H) labelled amino acid was digested with ribonuclease T1. The resulting oligonucleotides were separated with a DEAE-cellulose column and the distribution of radioactivities was analysed. The oligonucleotide bearing the radioactivity corresponded to the terminal oligonucleotide which was originally adjacent to the first guanylic acid from the terminal in the transfer RNA molecule. The terminal sequences in transfer RNA beyond the CCA-terminal were different for each amino acid, and in some cases, for instance yeast valyl RNA or leucyl RNA, at least two kinds of terminal sequence were observed. These sequences were also specific for each organism; for example, the structures near the CCA-terminal of valyl RNA of yeast and rat were different from each other, and those of the leucyl RNA of yeast, rat and Escherichia coli were distinguishable. Some amino acids were incorporated into transfer RNA by the enzymes from other organisms. When an amino acid was incorporated into transfer RNA by heterologous enzymes, the terminal nucleotide sequences near the attached amino acid were the intrinsic types for the transfer RNA. The observed results strongly suggest that the enzyme-recognizing site in the transfer RNA molecule is not located near the amino acid-accepting terminal.

The methyl groups in ribosomal RNA from Escherichia coli

Abstract 1. (1) Escherichia coli cells were labeled with [ Me - 14 C]methionine and [ 3 H]adenosine in the presence or absence of chloramphenicol. The ratio of 14 C to 3 H in RNA was taken as a measure of the extent of methylation of the RNA molecules. Methylation of mature 16-S ribosomal RNA (rRNA) was about 60 % higher than that of mature 23-S rRNA. The degrees of methylation of nascent 23-S and nascent 16-S rRNA formed in the presence of chloramphenicol were 56 and 12 % of the corresponding values for mature 23-S and 16-S rRNA, respectively. The methylation of transfer RNA (tRNA) was not significantly inhibited in the presence of chloramphenicol. 2. (2) Paper autoradiograms of nucleotides produced by alkaline hydrolysis of Me - 14 C-labeled RNA revealed a significant difference between mature 23-S and 16-S rRNA. The distribution of methyl groups in nascent 23-S (or 16-S) rRNA from chloramphenicol particles was qualitatively similar to that in mature 23-S (or 16-S) rRNA. Methylation of certain component nucleotides of the rRNA was found to be more or less selectively arrested in the presence of chloramphenicol. The autoradiogram of hydrolysates of tRNA was very different from that for rRNA. 3. (3) Chromatographic analyses on DEAE-cellulose columns of alkaline hydrolysates of Me - 14 C-labeled rRNA and tRNA suggested that the methyl content of the sugar moieties represented not more than 25 % of the total methyl groups in each RNA species.

Photochemical modification of transfer RNA and its effect on aminoacyl RNA synthesis

Transfer RNA was illuminated with visible light in the presence of methylene blue or the carcinogen 4-nitroquinoune-N-oxide. This procedure caused the loss of ultraviolet absorption of the guanine moiety. Analysis of base composition showed a decrease in guanine content, but no change in the other base components. The modification proceeded more rapidly in water than in buffer solution. The modification of polynucleotides proceeded slower than that of oligo- or mononucleotides. A scission of the backbone of the polynucleotide chain did not occur by this modification, but the configuration of tRNA was greatly altered. The guanine-modified tRNA was resistant to RNase T1 digestion, whereas it was digested by pancreatic RNase IA, RNase T2 or by alkali, as for the case of native tRNA. Amino acid-accepting ability of tRNA was destroyed with time of illumination. The inactivation rate varied for each amino acid. A significant decrease in the activity of proline acceptance was observed. This result supports the hypothesis that the anticodon sequence in tRNA would function in the recognition of an aminoacyl-RNA synthetase as well as in the decoding of messenger RNA.

Studies on nucleic acids of living fossils: I. Isolation and characterization of DNA and some RNA components from the Brachiopod Lingula

Abstract An RNA preparation from a living fossil, the Brachiopod Lingula, was fractionated by DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Five RNA components were separated: 5-S RNA; 4-S RNA which contains some minor bases and can accept an amino acid; a small RNA consisting of about 50 nucleotides; a larger RNA having a molecular weight of about 1 ·; 10 5 ; high-molecular-weight ribosomal RNA. Lingula DNA is a double-stranded DNA, of which the G+C content is 35.0 %.