THE BIOCHEMISTRY OF PLANT VIRUSES 97 



of flow of the solutions. An interesting observation was that much of the 

 nucleic acid was lost when the virus suspensions were precipitated with 

 ammonium sulfate, but as it now appears that the preparations were not 

 pure and were contaminated with nucleic acid-containing particles of plant 

 origin, it is not altogether certain whether the two phenomena are 

 coimected. 



The status of the tobacco ringspot virus remained much the same mitil 

 quite recently when the virus was reexamined by Steere (1956). Steere fomid 

 that the most suitable source of virus for purification was the cotyledons 

 of the Caserta squash {Cucurbita pepo), which yield some 45 mg. of virus 

 per hter of expressed sap. He also introduced a novel method for the clarifica- 

 tion of the sap, in which he used a chloroform-n-butanol treatment, a treat- 

 ment similar to that used by Morton (1953) in his experiments on extracting 

 enzymes from lipoid-containing materials. The aqueous phase so obtained 

 was subjected to three successive sedimentations at high speed out of 0.01 M 

 phosphate buffer of pH 7. The centrifuge pellets were resuspended in water 

 before the phosphate was added, however, because the pellets do not re- 

 suspend readily in the buffer. Controls run using healthy plants did not 

 yield any of the material. 



A. Physical Properties 



Solutions of the virus containing 2 % or more, when stored at 4°C. in 

 phosphate buffer (0.1 or 0.01 M) gradually deposit small crystals, which 

 evidently belong to the cubic system. Some appear to be derived from 

 octahedra, others from dodecahedra. In the crystalline state the virus 

 gradually loses its infectivity. 



Examined under the electron miscroscope, the ringspot virus particles 

 appear to be polyhedral in form, about 26 mja in diameter, especially if 

 freeze-dried, and show both hexagonal and pentagonal peripheries, indi- 

 cating that the particles are probably icosahedral in form (Kaesberg, 

 1956). 



As with most other plant viruses the number of particles required to 

 form a single local lesion (in cowpeas) is large, being of the order of 10^, and 

 as the preparations contain some 5 % of material having a lower sedimenta- 

 tion coefficient (89 *S) than that of the remainder (116 S), experiments were 

 carried out to check that the infectivity was really associated with the bulk 

 of the material. This was found to be so. Oddly enough the virus preparation 

 had only one electrophoretic component (11.3 X 10~^/cm./sec. /volt/cm. at 

 pH 7 in 0.05 M phosphate). This would suggest that the more slowly sedi- 

 menting component was similar to the nucleic acid-free virus proteins found 

 in other virus preparations. 



VOL. II — 7 



