FRAENKEL-CONRAT AND WILLIAMS 



and 52 |.ig./ml., respectively.'^ When 

 0.005 per cent TMV was held for sev- 

 eral hours in 5 per cent protein or 0.5 

 per cent nucleic acid, it was found 

 fully active upon dilution and assay. 

 This strongly suggests that the nonin- 

 fectivity of the two components can- 

 not be attributed to inhibition phe- 

 nomena (Table 1). 



267 



When each of the two components 

 was diluted for assay to 0.2 and 0.02 

 mg./ml., respectively, prior to mixing, 

 no activity was generated. Also, when 

 the complete reaction mixture (5-10 

 mg./ml.) was diluted to 0.1 mg./ml. 

 one minute after addition of the buffer 

 which triggers the aggregation, no ac- 

 tivity was obtained. About one hour 



Table 1 



Activity of Three Preparations of Reconstituted Virus and of TMV at 

 Different Assay Levels; Absence of Inhibitors from Components 



Material Tested 

 Reconstituted virus: 

 25,000-rpm. pellet 



40,000-rpm. pellet 

 TMV stock preparation 



TMV-protein added at 1000:1 

 TMV-RNA added at 100:1 



* Each figure is the average of 10 equivalent half-leaves. The 3 columns in top half 

 of table refer to separate preparations. 



t This is one of the preparations which appeared to increase in activity during storage. 



t Average and range of 9 assays (10 half-leaves each) of one TMV preparation over 

 3 months. 



at room temperature appears to be re- 

 quired for the formation of any active 

 rods. These experiments represent con- 



9 The usual assay method yielded usually 

 0.0, but at times up to 0.4, lesions per half- 

 leaf when phosphate alone was applied. This 

 result is probably due to accidental contami- 

 nation by active material applied to other 

 sites of the plant. Therefore, separate plants 

 were used when the absence of active virus 

 was in question. Standard TMV was applied 

 to one bottom leaf and the unknowns to all 

 others. This gave consistently 0.0 lesions with 

 the components used in virus reconstitution, 

 except when the nucleic acid was applied at 

 200 [ig./ml., twenty times the amount present 

 at the highest assay level of the reconstituted 

 virus (0.3 lesions per half-leaf). 



vincing control data; they also seem 

 to prove that we are dealing with a 

 definite chemical reaction and to ex- 

 clude most other interpretations. The 

 observation that the nucleic acid 

 gradually loses most of its activity dur- 

 ing several weeks of storage at 3° (in 

 aqueous solution, pH 5) also serves 

 as a control experiment, indicating the 

 crucial nature of its physical state. 

 Treatment of the nucleic acid (600 

 fxg.) with ribonuclease (0.2 \ig.) in 

 lO""* M magnesium sulfate rendered it 

 unable to combine with the protein to 

 produce active rods; also, substitution 

 of other nucleic acids (DNA from 



