INFECTIVITY OF TOBACCO MOSAIC VIRUS 449 



of 0.02 to 0.1 M salts at room temperature or upon incubation, but to be very 

 much more stable in 0.001 M or lower concentrations of salts. This degrada- 

 tion was evident from infectivity tests, from the decrease in sedimentability 

 as determined in 0.02 31 salts at 0° C, and finally from the appearance of 

 material that was dialyzable and not precipitated by 67 % ethanol (Fraenkel- 

 Conrat and Singer, 1958a; Reddi and Knight, 1957). 



The variability in the rate of this degradation has been very great with 

 different preparations, and even occasionally with the same preparation 

 upon repeated tests, as well as in the comparative rates of inactivation at 

 pH 5 and 7. Actually some preparations have been found which showed no 

 definite loss in infectivity after several hours of incubation in 0.1 M salts, 

 others which became completely inactive in pH 5 phosphate (0.1 M) but 

 remained unchanged at pH 7, wlide the majority of the samiples, lost over 

 90 % of the infectivity in one hour at 36°C. at any pH and in any 0.1 M salt, 

 except pyrophosphate. Whenever tested, sedimentation tests showed a 

 parallel behaviour, i.e., in experiments ui which no loss of activity was 

 observed there was also no decrease in sedimentability, while another nucleic 

 acid preparation under the same conditions showed both. 



The singular position of 23}Tophosphate was remarkable in that degrada- 

 tion, as measured by all criteria, was greatly retarded by this anion, as 

 compared with others. Thus, little loss of infectivity was observed upon in- 

 cubation for one hour in 0.1 M pyrophosphate of pH 7, even vrith preparations 

 that were completely inactivated in phosphate under the same conditions 

 (Fraenlcel-Conrat and Smger, 1958a). The mechanism of this effect is not clear. 

 It seems, however, quite probable that this is the reason why pyrophosphate 

 was found, as previously stated, a favorable medium for the reconstitution 

 reaction, yielding more rehably active virus preparations. It would seem that 

 the high ionic strength required for extensive reconstitution makes the nucleic 

 acid more susceptible to attack by trace enzymes prior to its incorporation 

 into virus rods, and that this competing degradative reaction is minimized by 

 the pyrophosphate medium. 



The mechanism of this degradative reaction is naturally of great import- 

 ance, particularly because its understanding may lead to its prevention. In 

 general, sensitivity to salts is not a property of nucleic acids. DNA is actually 

 sensitive to the absence of salts. The variabihty in the rate of degradation 

 suggests that we are deahng with a variable trace contaminant. In view of 

 the kno^\^l sensitivity of the infectious RNA to enzymes, it would appear most 

 bkely that traces of ribonucleases of plant and/or possibly bacterial origin 

 could account for the observed phenomena. It is known that the enzymatic 

 activity of ribonucleases is dependent on the ionic strength of the medium. 

 Particularly, when the enzyme concentration is hmiting, a dependence of its 

 efficiency on the salt concentration is to be expected. That this is in fact 



