FRAENKEL-CONRAT AND WILLIAMS 



protein subunits of a molecular weight 

 near 18,000 are arranged in a helical 

 manner to form a rod with a hollow 

 core. The nucleic acid is believed to 

 occur as strands in the core. Electron 

 micrographs which support this con- 

 cept have been obtained of the virus 

 at various stages of disaggregation.^"^'' 

 A protein isolated from infected plants 

 has been found to reaggregate— first to 

 short pieces of the presumed helix ly- 

 ing on end and resembling disks with 

 central holes and then to much longer, 

 but inactive, rods of the diameter of 

 the virus vet free from nucleic acid.^ 

 It has now been possible to achieve the 

 co-aggregation of inactive virus pro- 

 tein subunits and inactive virus nucleic 

 acid to give nucleoprotein rods which 

 appear to be infective. 



Preparation of Frotein and Nucleic 

 Acid Components— TMY was dia- 

 lyzed against pH 10-10.5 glycine 

 buffer (0.01 M) or pH 10.5 carbonate- 

 bicarbonate (0.1 M) at 3° C. for 48-72 

 hours. Undegraded virus was separated 

 bv cold ultracentrifugation, and the 

 supernatant was brought to 0.4 satura- 

 tion with ammonium sulfate. The pro- 

 tein alone precipitates (optical den- 

 sity 26o m/x/optical density28o mfi = -R = 

 0.65), leaving only nucleic acid (R = 

 2.0) in the supernatant; if this separa- 

 tion is not clean, longer alkali treat- 

 ment is necessary. The protein moiety 



2 Fraenkel-Conrat, H., and Singer, B., /. 

 Am. Chem. Soc. 76:180, 1954. 



3 Schramm, G., Schumacher, G., and Zillig, 

 W., Nature 175:549, 1955. 



4 Rice, R. v., Kaesberg, P. and Stahmann, 

 M. A., Bioch'mi. et biophys. acta 11:337, 1953. 



5 Hart, R. G., these Proceedings 41:261, 

 1955. 



^Takahashi, VV. N., and Ishii, M., Nature 

 169:419, 1952; Delwiche, C. C, Newmark, 

 P., Takahashi, W. N., and Ng, M. J., Bio- 

 cbivi. et biophys. acta 16:127, 1955; Com- 

 moner, B., Yamada, M., Rodenberg, S. D., 

 Wang, F. Y., and Easier, E. Jr., Science 118: 

 529, 1953. 



265 



is precipitated twice more with 0.25- 

 0.35 saturated ammonium sulfate, dia- 

 lyzed, brought to pH 7.0-8.0 with 

 NaOH, and finally again freed from 

 heavy particles, such as undegraded 

 virus, by ultracentrifugation. The pro- 

 tein gives a water-clear solution at 

 pH 7; the masked— SH group is still 

 present. The spectrum resembles that 

 of a mixture of tryptophan, tyrosine, 

 cysteine, and phenylalanine, simulating 

 the composition of the protein, al- 

 though the minimum (at 250 m^i) is not 

 quite as low (max./min. = 2.4 versus 

 2.9) (Fig. 1); P analyses (0.01-0.03 

 per cent) indicate removal of about 

 95-98 per cent of the nucleic acid. 

 Evidence for the absence of detectable 

 virus particles will be discussed below. 



The nucleic acid fractions from such 

 alkali-degraded TA4V are not as effec- 

 tive for reconstitution as that obtained 

 by the detergent method. - A virus so- 

 lution (1 per cent) containing 1 per 

 cent sodium dodecyl sulfate is adjusted 

 to pH 8.5 and held at 40° for 16-20 

 hours. Following this treatment, am- 

 monium sulfate is added to 0.35 satura- 

 tion, and the protein precipitate is 

 separated by centrifugation. When the 

 supernate is refrigerated, from 60 to 

 90 per cent of the nucleic acid precip- 

 itates {R = 2.0) and is centrifuged off 

 the next day, it is further purified by 

 repeated resolution in ice water and 

 precipitation with two volumes of cold 

 ethanol and a few drops of 3 M pH 

 5 acetate. The nucleic acid solution is 

 finally subjected to cold ultracentrifu- 

 gation to remove any traces of virus. 

 The virtual absence of protein is indi- 

 cated by a minimum in O.D. near 230 

 m\x (max./min. = 3.0) (Fig. 1). 



In a few preliminary ultracentrifuge 

 experiments, kindly performed by Dr. 

 Howard K. Schachman, the nucleic 

 acid preparations exhibited one prin- 

 cipal boundary with a sedimentation 



