C.A. Knight

An oligonucleotide mapping procedure and its use in the study of tobacco mosaic virus nucleic acid

Abstract A two-dimensional procedure was developed employing paper electrophoresis and paper chromatography, which permitted the separation and identification of all major products obtained by digestion of tobacco mosaic virus ribonucleic acid (TMV-RNA) with pancreatic ribonuclease. The map so obtained showed nineteen spots accounting for approximately 90 % of the optical density of the material applied. The substances in the major spots were quantitatively estimated by elution and spectrophotometry, and qualitatively identified by application of standard procedures for determining the composition of nucleotides and oligonucleotides. The homogeneities of the isolated compounds were evaluated by elution and subjection of eluates to ion-exchange and paper-chromatographic procedures.

Protein synthesis in tobacco protoplasts infected with tobacco mosaic virus

Abstract A detailed study was made of protein synthesis in tobacco mosaic virus-infected tobacco protoplasts using the techniques of slab polyacrylamide gel electrophoresis and autoradiography. Three novel proteins not demonstrable in uninfected protoplasts were detected. Their molecular weights were estimated to be 17,500, 135,000, and 165,000. The smallest of these proteins is undoubtedly the viral coat protein but the identities of the other two were not established.

The ribonucleic acid, lipid, and polysaccharide constituents of influenza virus preparations

Abstract The ribonucleic acid, lipid, and polysaccharide constituents of three strains of influenza virus (PR8, DSP, and Lee) and of related host materials were investigated by chromatographic and spectroscopic techniques. Cephalin, sphingomyelin, lecithin, and unesterified cholesterol were found in the virus strains examined in approximately the same proportions as in the material derived from normal allantoic fluid or membrane. In addition, the proportions of phospholipid:cholesterol:triglyceride were of the same order in virus and normal membrane. All three strains were found to contain (as polysaccharides) galactose, mannose, fucose, and amino sugar in approximately the same proportions as in the material from allantoic fluid or membrane. From the ribose released on hydrolysis of the viruses, it was possible to estimate that the three strains of influenza virus contain about 0.8% ribonucleic acid.

Cyclic DNA of shope papilloma virus

Double-stranded DNA of Shope papilloma virus prepared for electron microscopy by the protein monolayer technique was found to exist in loop-like structures without ends. Mean contour lengths of such DNA ranged from 2·3 to 2·8 μ. The phenol-extracted material showed similar conformations whan the DNA was allowed to diffuse to a preformed monolayer of cytochrome c , and when a mixture of DNA and cytochrome c was spread on an aqueous surface. DNA diffusing from virus subjected to osmotic shock also appeared as rings, but the rings were less convoluted than those in the preparations extracted by phenol. The variation in length is tentatively attributed to varying degrees of local preparative denaturation in the DNA strands.

Structure of Shope papilloma virus particles.

Abstract Shope papilloma virus from warts of cottontail rabbits was partially purified by differential centrifugation and then subjected to rate zonal centrifugation in sucrose or glycerol density gradients where the particles were separated into three bands and a small pellet. The top component consisted of particles that were of characteristic size for papilloma virus but, upon examination in the electron microscope, after treatment with phosphotungstic acid, most of them appeared to be hollow. This fraction also gave a protein-like absorption curve in the ultraviolet, contained very little phosphorus, and was almost noninfectious. By contrast the particles in the middle band appeared completely formed, contained appreciable phosphorus, had a nucleoprotein-like absorption curve, and were highly infectious. It is not known whether the two types of particle are produced as such in the host or are a result of preparation. In all particles a pattern of spots was seen either over the surface or around the periphery. The spots were of uniform size and were arrayed in a definite pattern, suggesting that they represent a subunit structure.

A comparison of the ultraviolet-light inactivation of infectious ribonucleic acid preparations from tobacco mosaic virus with those of the native and reconstituted virus.

Abstract The quantum yield for the inactivation of infectious ribonucleic acid preparations (RNA) from tobacco mosaic virus (TMV) by ultraviolet light (UV) at 230, 248, 254, 265, and 280 mμ was found to be independent of wavelength. A dependence of quantum yield on concentration was observed, however. Photoreactivation of irradiated RNA was constant at all wavelengths. Therefore the absorption and action spectra of this RNA, with and without photoreactivation, coincide. The presence of small amounts of protein essential for RNA infectivity has not been excluded by this approach. The rate constants for the UV inactivation of TMV were not constant, but highest at 230 mμ and lowest at 280 mμ. Native and reconstituted TMV were equally sensitive to UV at all wavelengths tested.

Polar stripping of protein subunits from tobacco mosaic virus

Abstract Treatment of tobacco mosaic virus (TMV) with sodium dodecyl sulfate (SDS) at 37° leads to the progressive removal of protein subunits (“stripping”). Electron micrographs show that the protein is not removed from random sites on the rod, but rather from one end, resulting in a progressive shortening of the nucleoprotein rod and uncovering of the ribonucleic acid (RNA) strand. Treatment of partially stripped virus (PSV) with snake venom phosphodiesterase or polynucleotide phosphorylase leads to loss of infectivity at rates paralleling those of TMV RNA when treated similarly. On the other hand, spleen phosphodiesterase does not alter the infectivity of PSV under conditions in which TMV RNA is inactivated. When PSV labeled with C 14 is treated with pancreatic ribonuclease, C 14 -adenosine is released in stoichiometric amounts. It is concluded that the stripping of TMV by SDS occurs through the progressive removal of protein subunits from the 5′-linked end of the viral rod.

Are cucumber viruses 3 and 4 strains of tobacco mosaic virus?: A review of the problem

Abstract An attempt is made to deal with the questions of what is a virus strain, and which are the critical criteria of strain relationship, by examining specifically the relationship of cucumber viruses 3 and 4 to tobacco mosaic virus. In scrutinizing this relationship, various existing criteria are applied and three new chemical standards are introduced and applied. It is finally concluded that cucumber viruses 3 and 4 are probably not strains of tobacco mosaic virus.

The protein subunit of potato virus X

Abstract The N-terminal amino acid residue of the capsid protein of potato virus X is acetylated as it is in several other plant virus proteins. On the basis of amounts of acetic acid recovered from the N-terminal residue, and amino acid analysis coupled with peptide mapping, the protein subunit of potato virus X was found to consist of about 210 amino acid residues, each subunit having a molceular weight of approximately 22,300.

The peptide chains of some plant viruses.

Abstract Tomato bushy stunt and potato X viruses and cucumber viruses 3 and 4 were analyzed for amino-terminal and carboxyl-terminal amino acid residues. No free amino-terminal groups could be detected for any of these viruses using the fluorodinitrobenzene and phenylisothiocyanate methods. Using a hydrazinolysis procedure, the C -terminal residues of tomato bushy stunt, potato X, and the cucumber viruses were found to be leucine, proline, and alanine, respectively. A subunit size was calculated for each virus from the number of moles of terminal amino acid found for a known weight of virus.