ISOLATION AND COMPOSITION OF PENTOSE NUCLEIC ACIDS 377 



of the less-stable viruses which do not survive chemical fractionation pro- 

 cedures. The success of this method for the purification of plant viruses 

 depends on the absence of material of corresponding particle size and 

 stability in uninfected plants. A nucleoprotein of the size of plant viruses 

 has been isolated by Pirie'^ from the sap of young uninfected tobacco 

 leaves. However, the amount of this material in older leaves is very small, 

 and it is removed in the course of the procedures used to clarify the in- 

 fected sap prior to the sedimentation of the plant virus. Consequently, 

 plant viruses isolated from cell sap, clarified by precipitation with ethanol, 

 freezing, and exposure to inorganic phosphate, will not be contaminated 

 by the nucleoprotein of normal plant leaves.^® 



Several strains of tobacco mosaic and related viruses have been isolated 

 by fractional centrifugation for a study of the amino acid and nucleotide 

 composition of the virus nucleoprotein. ^^-^^ The preparation of cucumber 

 virus 4 (CV4) is given in detail. 



(1) Preparation of Cxicumber Virus 4 {CV^y-. Young cucumber plants were inocu- 

 lated with CV4 by rubbing one leaf on each plant with a gauze pad saturated with 

 infective juice. This juice was obtained from a cucumber plant showing the typical 

 yellow mottling produced by cucumber virus 4 in members of the Cucurbitaceae. 

 About 1 month after inoculation, the plants were harvested and placed in a room 

 kept at —12°. After a few days the frozen plants were ground. Three per cent by 

 weight of dipotassium phosphate in a 50% solution was mixed with the pulp, and 

 after about 2 hours the juice was expressed from the cold but completeh' thawed 

 pulp. The juice was passed through a celite filter to remove coarse particles of green 

 pigment and extraneous matter, and then the virus was sedimented in the form of 

 pellets by centrifugation at 20,000 to 30,000 r.p.m. for 30 minutes. The supernatant 

 liquid, which was practically free of virus, was discarded, and the pellets were dis- 

 solved in small amounts of distilled water. The combined solutions of virus pellets 

 were spun at about 3000 r.p.m. on an angle centrifuge for 30 minutes to remove green 

 pigment and insoluble colloidal matter. The supernatant liquid, which contained the 

 virus, was then returned to the high-speed centrifuge and the process was repeated 

 about three times, a longer period being allowed for the high-speed centrifugation as 

 the virus became more concentrated. In many cases it was found possible to effect a 

 better separation of green pigment from the virus by dissolving the pellets obtained 

 by the first two high-speed centrifugations in 0.1 M phosphate buffer at pH 7 and then 

 using distilled water as a solvent for the pellets of the last two centrifugations. All 

 of the preparations used for elementary analysis were further purified by dialysis 

 against flowing distilled water for 48 hours. 



Pellets of purified virus ranged from 0.1 to 0.4 g. per liter of expressed juice as 

 compared with 2-2.5 g. per liter ordinarily obtained for tobacco mosaic virus from 

 the juice of diseased turkish tobacco plants. 



'« N. W. Pirie, Biochem. J. 47, 614 (1950). 



'7 C. A. Knight and W. M. Stanley, /. Biol. Chem. 141, 29 (1941). 



18 C. A. Knight, J. Biol. Chem. 171, 297 (1947). 



1" C. A. Knight, J. Biol. Chem. 197, 241 (1952). 



