ISOLATION AND COMPOSITION OF PENTOSE NUCLEIC ACIDS 389 



Similar concentrations gave negative biuret tests. The yield of ribonucleic acids 

 varied from 20 to 30% of the total tissue ribonucleic acid. 



3. Isolation of PNA from the Tissues of Higher Plants 



The study of the nucleic acids of plant tissues has been rather neglected 

 in recent years. Kay and Dounce^^ reported the isolation of PNA from 

 wheat germ by a method analogous to that used for animal tissue, namely 

 by the use of sodium dodecyl sulfate. Their product is characterized by a 

 very low ratio of nitrogen to phosphorus and may be contaminated with 

 other phosphorus-containing compounds. So far, no analyses of the nucleo- 

 tide composition of preparations of plant PNA have been reported. On 

 the other hand, the nucleic acids of plant viruses have been isolated and 

 their composition and properties have been determined. The pentose 

 nucleic acids of several strains of tobacco mosaic virus and the related rib 

 grass viruses, were detached from protein by heating for 1 minute as 

 described by Cohen and Stanley," and modified by Knight. ^^ In the case 

 of turnip yellow mosaic virus, the protein was denatured and the PNA 

 liberated by treatment with 30% ethanol.^^ 



(1) Isolation of PNA from Strains of Tobacco Mosaic Virus and Cucumber Virus.^^ 

 Six cubic centimeters of 0.13 M sodium chloride, 0.001 M with respect to Sorensen's 

 phosphate (K2HPO4-KH2PO4 , 11:5) and at a pH of about 7, was heated in a 15-cc. 

 conical glass centrifuge tube in a water bath at 100°. To this was added 2 cc. of virus 

 solution at a concentration of 30 to 80 mg. of virus per cubic centimeter. The mixture 

 was stirred by being drawn up and down in a dropping pipet for about 15 seconds. 

 By this time the mixture had reached a temperature of about 100° and heating was 

 continued for 1 minute; the tube was then withdrawn and placed in an ice bath. The 

 contents of several tubes were usually pooled and spun at 7000 r.p.m. in an angle 

 centrifuge in order to remove coagulated protein. The clear supernatant fluid con- 

 taining the sodium nucleate was dialyzed overnight at 4° against 18 1. of flowing 

 distilled water in a Kunitz-Simms rocking dialyzer. The ultraviolet absorption at 

 260 m^ of the nucleate before and after dialysis was essentially identical, which 

 indicates that no significant quantity of dialyzable nucleic acid material was pro- 

 duced by the cleavage method or by subsequent dialysis. The dialyzed nucleate solu- 

 tion was concentrated to 0.07 to 0.02 of its volume by pervaporation, and the small 

 amount of insoluble matter appearing during concentration, together with small 

 quantities of soluble virus fragments, removed by centrifugation at 40,000 r.p.m. 

 (102,000 X g, average) for 1 hour in the No. 40.2 rotor of the Spinco model L centri- 

 fuge. The clear supernatant fluid was lyophilized and the residue was dried to con- 

 stant weight in a drying oven at 110° or in vacuo over P2O6 at 78°. 



Various modifications of the above procedure were tried with results which may 

 be summarized as follows. Lithium chloride can be substituted for sodium chloride. 

 If the final concentration of salt is about 0.1 M, coagulation of the denatured protein 

 seems to be greatly aided by the presence of a small amount of phosphate, but, if a 

 salt concentration in the neighborhood of 0.3 M is used, the phosphate is dispensable. 

 In tests with up to 1 M of sodium chloride, the yields of sodium nucleate were found 



« R. Markham and J. D. Smith, Biochem. J. 49, 401 (1951). 



