430 H. FRAENKEL-CONRAT 



is an intriguing observation (Frisch-Niggemeyer, 1956). The main architec- 

 tural relationship of the viral components also appears to be constant. For 

 in all cases that have been studied, the protein furnishes an outer shell for 

 the more internally located nucleic acid. This appears to be true for the 

 spherical plant or animal viruses, for the rod-shaped plant viruses, and for 

 the tadpole-shaped bacteriophages. 



I. Purification of Plant Viruses 



Since the isolation of pure tobacco mosaic virus (TMV) by Stanley in 1935, 

 a number of plant viruses have been prepared in pure or near-pure state; 

 several of these were obtained in crystalline or paracrystalline form. The 

 chemical methods of isolation that enabled Stanley and others to achieve the 

 first instances of success (Stanley, 1935a; Bawden and Pirie, 1937a) have in 

 many instances been later replaced by the gentler physical methods — in 

 particular, by differential centrifugation and density gradient centrifugation. 

 As a typical example, the routine procedure for isolation of TMV in this 

 laboratory will be described. Other plant viruses have been purified by varia- 

 tions of the same procedures as dictated by specific peculiarities of the par- 

 ticular viruses or the host plant material. The references listed in Table I 

 will direct the reader to these procedures. 



A. Procedure for the Preparation of TMV 



Turkish tobacco leaves are harvested three weeks after inoculation with 

 TMV. They are frozen in a deep-freeze and may be stored at this stage. 

 They are then ground in a meat grinder, mixed with a concentrated solution 

 of K2HPO4 (3 gm. per 100 gm. leaf material), and allowed to melt. After 

 centrifugation in a basket centrifuge, the juice is subjected to ultracentri- 

 fugation in the Model G Spinco ultracentrifuge (30-rotor, 22,000 r.p.m. for 

 1 hour). The pellet is dispersed in 0.1 M (pH 7) phosphate, clarified by centri- 

 fugation for 10-15 minutes at 4000-8000 r.p.m. and again ultracentrifuged. 

 After about two more cycles of such alternate high- and low-speed centri- 

 fugation in 0.1 M phosphate, and two with water as solvent, the final pellet 

 is dispersed in water and sterilized by filtration. The use of chloroform- 

 saturated solvents throughout appears to be advantageous. 



A convenient technique for near quantitative isolation of TMV from a few 

 leaves or leaf discs was described by Schlegel and Rawlins (1953). It yields 

 within a few hours a spectrophotometrically clean virus solution. 



B. Isolation and Properties of Other Plant Viruses 



A few of the better characterized plant viruses are listed in Table I, to- 

 gether with some selected data concerning procedures of isolation, the ap- 

 pearance and composition of the purified viruses, and a few key references. 



