Tobacco Mosaic Virus Nucleic Acid Research Articles

Sorption of Water Vapour by Tobacco Mosaic Virus Protein and Nucleic Acid, and Photoinactivation of the Nucleic Acid

SUMMARY A hysteresis has been noted between the adsorption and desorption isotherms of TMV nucleoprotein. By comparison with hydrations of RNA and protein separately, the hysteresis can be attributed to TMV capillary water. Following the inactivation by ultraviolet irradiation of nucleic acid in solution, some photorecovery on bean leaves can be observed; this does not seem to occur if the RNA is irradiated dry.

The inhibitory action of mineral oil on the number of local lesions on Nicotiana glutinosa L. leaves inoculated with tobacco mosaic virus

The effect of oil on the number of local lesions produced by tobacco mosaic virus (TMV) in Nicotiana glutinosa L. leaves was studied. Application of oil before or after inoculation of the leaves affected the number of local lesions produced to the same extent. An equivalent inhibition was found when nucleic acid of TMV was used, indicating that uncoating of the virus is not sensitive to or influenced by oil. Oil treatment at different times after inoculation showed that the lowest number of local lesions was produced by the leaves with oil at 15 min after inoculation. The results suggest that not every infecting particle is sensitive to oil at the same moment. Repeated treatments at intervals of 15 min over a period of 2 hr caused an almost complete inhibition.

The inhibition, inactivation and precipitation of tobacco mosaic virus nucleic acid by components of leaf extracts

Unless calcium is removed from leaf extracts containing tobacco mosaic virus nucleic acid, infectivity measurements are invalid. In some circumstances, calcium, nicotine and spermine can prevent the movement of nucleic acid into the water phase when extracts are made in the presence of excess phenol. The concentrations needed for this effect are greater than those usually found in leaf extracts, but it is possible that association of the virus with these sub­stances produces local concentrations large enough to influence the infectivity of extracts.

Factors affecting the amount of tobacco mosaic virus nucleic acid in phenol-treated extracts from tobacco leaves

Extracts from the lower leaves of tobacco plants infected with tobacco mosaic virus and pulped in the presence of phenol are less infective than extracts from leaves pulped and then treated with phenol. Extracts from uninfected leaves mixed with purified virus behave similarly. The difference becomes progressively smaller as leaves are taken from further up the plant. The difference is smaller when the interval between pulping and adding phenol is short, and it is still smaller if air is rigidly excluded during the interval. Fixation of virus nucleic acid to the leaf fibre is the main factor responsible for this difference. Part of the normal nucleic acid of the leaf is fixed similarly. Fixation is partly prevented by including yeast nucleic acid in the extraction fluid, or by excluding Ca 2+ from it by adding citrate. Part of the nucleic acid is held tenaciously by the fibre. The possible significance of this fixation in vivo in controlling the apparent susceptibility to infection of plants in different physiological states is discussed.

Conformational changes induced in tobacco mosaic virus nucleic acid by ultraviolet radiation

In order to study any Conformational changes associated with ultraviolet irradiation of TMV-RNA, methods of circular dichroism and absorbance-temperature profile were applied. RNA irradiated in water at 254 nm showed a distinct change in CD spectrum, but there was only a slight change accompanying irradiation in phosphate buffer.

Sensitivity of tobacco mosaic virus to ultraviolet irradiation.

Japanese Journal of MicrobiologyVolume 16, Issue 5 p. 441-443 ArticleFree Access Sensitivity of Tobacco Mosaic Virus to Ultraviolet Irradiation Manabu Umekawa, Manabu Umekawa Institute for Plant Virus Research, Chiba, 280Search for more papers by this authorNobuyuki Oshima, Nobuyuki Oshima Institute for Plant Virus Research, Chiba, 280Search for more papers by this author Manabu Umekawa, Manabu Umekawa Institute for Plant Virus Research, Chiba, 280Search for more papers by this authorNobuyuki Oshima, Nobuyuki Oshima Institute for Plant Virus Research, Chiba, 280Search for more papers by this author First published: May 1972 https://doi.org/10.1111/j.1348-0421.1972.tb00681.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES 1Conrat, H. Fraenkel 1957. Degradation of tobacco mosaic virus with acetic acid. Virology 4: 1– 4. 2Conrat, H. Fraenkel, and Singer, B. 1959. Reconstitution of tobacco mosaic virus. III. Improved methods and the use of mixed nucleic acids. Biochim. Biophys. Acta 33: 359– 370. 3Funatsu, G. 1967. Studies on chemical structure of tobacco mosaic virus-protein. J. Agr. Chem. Soc. Japan 41: R53– R 63. 4Haschemeyer, R., Singer, B., and Conrat, H. Fraenkel 1959. Two configurations of tobacco mosaic virus-ribonucleic acid. Proc. Nat. Acad. Sci. U. S. 45: 313– 319. 5Nozu, Y., Ohno, T., and Okada, Y. 1970. Amino acid sequences of some common Japanese strains of tobacco mosaic virus. J. Biochem. 68: 39– 52. 6Oshima, N., Ohashi, Y., and Umekawa, M. 1971. Studies on some strains of tobacco mosaic virus pathogenic to crucifer plant. 1. Physical and chemical studies. Ann. Phytopath. Soc. Japan 37: 319– 325. 7Siegel, A., and Wildman, S. G. 1954. Some natural relationships among strains of tobacco mosaic virus. Phytopathology 44: 277– 282. 8Siegel, A., and Wildman, S. G. 1956. Sensitivity of ultraviolet light of infectious tobacco mosaic virus nucleic acid. Nature 178: 1117– 1118. 9Streeter, D. G., and Gordon, M. P. 1967. Ultraviolet photoinactivation studies on hybrid viruses obtained by the cross-reconstitution of the protein and RNA components of U(l) and U(2) strains of TMV. Photochem. Photobiol. 6: 413– 421. Citing Literature Volume16, Issue5May 1972Pages 441-443 ReferencesRelatedInformation

Conformational studies of TMV-RNA: Calorimetric and related investigations

Calorimetric measurements of heats arising from transferring Tobacco Mosaic Virus Nucleic Acid (TMV-RNA) from a low to high ionic strength value show that such transfers result in considerable amounts of exothermic heat. Furthermore, the heats measured were found to increase with increasing ionic strength. Velocity sedimentation and viscosity measurements show that in solutions of ionic strength greater than 0.1, the sedimentation coefficient and specific viscosity values of TMV-RNA remain practically unchanged, suggesting that changes in molecular size and hydrodynamic characteristics cannot fully account for the observed heat effects. Optical Rotatory Dispersion (ORD) and Circular Dichroism (CD) measurements of TMV-RNA solutions were made as a function of ionic strength. These studies show that the structure increases with ionic strength. Therefore, conformational changes, mostly as a result of increase in base-pairing and possibly to a smaller extent as a result of changes in base-stacking, appear to be responsible for most, if not all, of the observed heat effects. Energy calculations using the calorimetrically measured heat values show that the energy associated with the conformational changes in TMV-RNA arising from a change in ionic strength from 0.01 to 0.8 is about −2 kcal/mole and this is of the right order of magnitude.

Quantum yields for inactivation of tobacco mosaic virus nucleic acid by ultraviolet radiation (254 nm.).

SUMMARY The quantum yield, under non-photoreactivating conditions, for inactivation of tobacco mosaic virus RNA by ultraviolet radiation (254 nm.) is independent of the RNA concentration but varies appreciably with the ionic strength of the irradiation solvent, being approximately three times greater in water than in 0.1 m-potassium phosphate buffer. Photoreactivation of inactivated tobacco mosaic virus RNA is dependent on RNA concentration, ionic strength of solutions during irradiation, and on the light quality used for photoreactivation. These data can be interpreted as evidence that pyrimidine hydrate is a lethal lesion at least in RNA irradiated at low ionic strength.

Comparative photoreactivation of ultraviolet light-inactivated tobacco mosaic virus ribonucleic acid on Chenopodium, pinto bean, and tobacco plants

Tobacco mosaic virus nucleic acid (TMV-RNA), inactivated by ultraviolet light, 2537 A, was photoreactivated on leaves of Chenopodium, Pinto bean, and tobacco. There was greater photoreactivation on tobacco leaves than on the leaves of the other two plants. The quantum yield for the inactivation of the nucleic acid was about the same regardless of which plant was used as host when leaves were infected and left in the dark. This finding indicates the lack of dark recovery mechanisms in plant leaves for repair of TMV-RNA inactivated at 2537 A. Illuminated leaves had higher temperatures than leaves left in the dark. This temperature difference did not influence the extent of photoreactivation, nor did the presence of bentonite in the inoculum.

Relation between incorporation of 2-thiouracil into tobacco mosaic virus nucleic acid and virus inhibition

When tobacco mosaic virus (TMV) infected tobacco leaves were floated in nutrient solutions containing 2-thiouracil (2-TU), the analog which was incorporated into the virus ribonucleic acid (RNA) was present only as 2-thiouridylic acid; no 2-thiouridine or 2-thiouridine 3′(2′),5′-diphosphate was detected in the TMV-RNA preparations. No differences in TMV-RNA containing 2-TU and normal RNA could be observed on examination in the analytical ultracentrifuge. Significant intercorrelations were found between 2-TU incorporation into TMV-RNA, the loss of infectivity, and reduction in virus yield.

Competition between infectious tobacco mosaic virus nucleic acid and intact virus on Nicotiana glutinosa.

PREVIOUS work by Wu and Rappaport1 was concerned with the phenomenon of competition between intact strains of tobacco mosaic virus. In each case, a mild strain of tobacco mosaic virus (U2 or VM) was used to interfere with the establishment of infections by the common strain, U1. In this communication, competition between these same strains but involving infectious ribonucleic acid (RNA) and intact virus will be described.

Photo-reactivation of tobacco mosaic virus in vivo.

TOBACCO mosaic virus, inactivated in vitro by ultra-violet irradiation, could not be photo-reactivated by exposing plants, inoculated with the inactivated virus, to light1,2. However, ribonucleic acid preparations from tobacco mosaic virus could be photo-reactivated in this manner, following inactivation by ultra-violet light1,3. Siegel et al.4 investigated the effects of ultra-violet light on tobacco mosaic virus and tobacco mosaic virus nucleic acid, at different times after inoculation of Nicotiana glutinosa plants, on which local necrotic lesions are formed. They interpreted their results to suggest that the nucleic acid of tobacco mosaic virus is freed from its protein moiety during a lag period of approximately 2.5 hr. following inoculation and before the infective centre begins to increase in resistance to ultra-violet light. If this model is correct, and the nucleic acid of tobacco mosaic virus separates from the protein, then it should be possible to photo-reactivate the nucleic acid during the period when the separation occurs.

Use of snake venom phosphodiesterase in place of pancreatic ribonuclease for the testing of nucleic acid preparations from tobacco mosaic virus.

DEPROTEINIZED plant virus preparations are customarily incubated with pancreatic ribonuclease in order to show that their infectivity is not due to residual virus particles. With tobacco mosaic virus nucleic acid, exceedingly small concentrations of the enzyme (10−8 mgm./ml.) are sufficient to abolish infectivity after incubation for 24 hr. at room temperature, whereas tobacco mosaic virus is not affected by a thousandfold higher concentration1. However, under these conditions tobacco mosaic virus nucleic acid incubated without enzyme often loses most or all of its activity1. Shorter times of incubation necessitate the use of higher ribonuclease concentrations, but at higher concentrations pancreatic ribonuclease, when mixed with plant viruses, inhibits infection2–4. This effect occurs immediately after ribonuclease is mixed with the virus and does not increase upon incubation of the mixture2. Consequently, as the ribonuclease concentration is increased and the incubation period shortened, the range within which tobacco mosaic virus nucleic acid is inactivated and tobacco mosaic virus is not affected becomes progressively narrower. If one considers that the effect on the virus of a given concentration of ribonuclease is furthermore a function of salt concentration and of concentration of tobacco mosaic virus2, it becomes evident that the results of incubation studies with ribonuclease must be carefully evaluated before definite conclusions can be drawn.

Distribution of guanylic acid residues in tobacco mosaic virus nucleic acid.

THE mode of arrangement of purine and pyrimidine nucleotides in tobacco mosaic virus nucleic acid was studied using the actions of pancreatic ribonuclease and micrococcal phosphodiesterase. This work showed that in tobacco mosaic virus nucleic acid the purine nucleotides and pyrimidine nucleotides occur in ‘clusters’ of two or more and also ‘singly’ alternating with each other. The extension of these studies to nucleic acids of three viral strains (TMV, HR and M) revealed for the first time significant differences in the arrangement of nucleotides in each of their molecules1–5. Additional information on the distribution of guanylic acid residues in tobacco mosaic virus nucleic acid, obtained by the action of an enzyme specific to secondary phosphate esters of guanosine 3′-phosphates, is given in the present communication.

Mode of action of micrococcal phosphodiesterase.

IT was recently reported that phosphodiesterase isolated from Micrococcus pyogenes var. aureus hydrolyses tobacco mosaic virus nucleic acid giving rise to di- and mono-nucleotides1,2. On the basis of its action on this acid and synthetic polymers, polycytidylic acid and polyuridylic acid, it was postulated that the enzyme first cleaves the large nucleic acid molecule into several polynucleotides, which are further broken down by stepwise cleavage of mononucleoside 3′-phosphates starting from one end, until they are reduced to the size of dinucleotides, which are resistant to further action of the enzyme. Thus the enzyme appears to have both endo- and exo-actions. From the amounts of dinucleotides present in the final digest of tobacco mosaic virus nucleic acid, one can calculate the number of intramolecular breaks that are produced in the acid by the endo-action of the enzyme, by assuming that the dinucleotides are at one end of the resultant polynucleotide fragment. In these calculations the data used for the purine and pyrimidine composition of tobacco mosaic virus nucleic acid are those published earlier3, and the molecular weight of nucleic acid was taken as 2 × 106 (ref. 4). From the calculations it appears that tobacco mosaic virus nucleic acid has been degraded to approximately 1,240 polynucleotide fragments, having an average weight of 1,600 or an average length of approximately 5 nucleotides.

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

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.

Serological studies with X protein, tobacco mosaic virus, polymerized X protein, and virus reconstituted from nucleic acid and X protein

Anti-TMV and anti-X protein sera cross-react with X protein and tobacco mosaic virus (TMV), respectively. The potency of the X protein antigen is greatly enhanced when precipitin reactions are carried out at pH 5.3. In accordance with the results of Starlinger, anti-TMV serum absorbed with X protein still reacts with TMV. Anti-X protein serum absorbed with TMV, however, is inactive against X protein. Anti-TMV serum absorbed with X protein will react with X protein if it is polymerized into rod-shaped particles or if it is reconstituted into virus with TMV nucleic acid. But anti-TMV sera absorbed with polymerized X protein or with TMV reconstituted from X protein and virus nucleic acid do not react with TMV.

Partition cell analysis of infective tobacco mosaic virus nucleic acid

The infective principle of ribonucleic acid (RNA) isolated from tobacco mosaic virus (TMV), either by Duponol C or water-saturated phenol, has a sedimentation constant of 19·6 Svedberg units at 5°C in 0·02 M -phosphate, pH 7·3, as determined by partition cell ultracentrifugal analysis. This corresponds to an S 20 of 30. In fresh samples of RNA, the bulk of the material has the above sedimentation rate. The weight average molecular weight of such samples is 2 × 10 6 as determined by light scattering. The sedimentation rate of the infective principle in samples degraded at room temperature to various fractions of residual activity remains the same, although the bulk of the material sediments at a much slower rate. It is therefore concluded that the smaller components do not carry a detectable amount of the infectivity.

Degradation of tobacco mosaic virus nucleic acid with micrococcal phosphodiesterase

The phosphodiesterase present in the culture medium of Micrococcus pyogenes var. aureus was purified and its action on tobacco mosaic virus nucleic acid was studied. In the complete digest nucleoside 3′-phosphates of both purines and pyrimidines and also several di-nucleotides were found. Its action on polyuridylic and cytidylic acids also resulted in the production of the corresponding nucleoside 3′-phosphates and dinucleotides. The enzyme was found to have no action on purine or pyrimidine cyclic nucleotides. The possible mode of its degradation of tobacco mosaic virus nucleic acid was discussed.

Studies on tobacco leaf ribonuclease III. Its role in the synthesis of tobacco mosaic virus nucleic acid

Ribonuclease activity in tobacco leaves was found to vary with the age of leaf. The old bottom leaves were found to contain more RNase than the top young leaves. The number of lesions apprearing on the leaves and also the amount of tobacco mosaic virus synthetised following infection with TMV corresponded with the RNase content of the leaf. There was a significant increase in the RNase content of Turkish tobacco leaves following infection with TMV. The significance of these findings and the possible role of RNase in the synthetic processes were discussed.