440 H. FRAENKEL-CONRAT 



with virus protein luider the favorable conditions to be discussed below gives 

 virus-like rods of full virus infectivity in 30-80 % yield (Fraenkel-Conrat, 

 1957c; Fraenkel-Conrat and Singer, 1958b). Thus the doubts mentioned at 

 the end of the pre\dous section are deprived of their basis, and the nucleic 

 acid as isolated by either of two procedures is shown to be potentially almost 

 fully infective. Eeconstitution is thus the best method of functionally testing 

 nucleic acid preparations, and is routinely used for this purpose in our labora- 

 tory. Other important uses of this reaction will be discussed in two subse- 

 quent sections. 



The high efficiency of reconstitution now often attained has been the result 

 of various changes of the original procedure. At present, the nucleic acid is 

 added at room temperature to a 0.1 % solution of the protein in 0.1 M pyro- 

 phosphate (tetrasodium pyrophosphate adjusted with HCl to pH 7.0). For 

 preparative purposes an approximately equivalent amount of nucleic acid 

 is used, i.e., one-fifteenth to one-twentieth of the protein. Slightly higher 

 relative yields in reconstituted infectivity are often obtained if relatively less 

 nucleic acid (e.g., 1/100 or 1/200 of the protein) is used. For most nucleic 

 acid preparations the use of pyrophosphate is crucial, while with an occasional 

 sample a phosphate medium wiU give a similar extent of reconstitution (see 

 later for discussion of the role of pyrophosphate). Lower salt concentrations 

 and variations in pH below 6.5 or above 8.0 give distinctly lower yields. 



Reaction mixtures containing reconstituted virus generally are directly 

 tested for infectivity after suitable dilution, and the yield is calculated on 

 the basis of the nucleic acid in the reaction mixture compared with the 

 nucleic acid content of the standard virus, wliich is assumed to represent 

 5 % of its weight. Naturally, the total weight of the two viral components 

 can be used and compared to the weight of standard virus when these are 

 used in equivalent proportions (protem/ENA=20). The reaction can be 

 terminated at any stage by dilution, preferably by simultaneous addition of 

 ribonuclease (1 % of virus potentially present). This enzyme does not attack 

 or inhibit TMV or reconstituted TMV when used at such concentrations, but 

 it quickly degrades any free nucleic acid. The time required for the appear- 

 ance of maximal infecti\'ity appears to be about 6 hours in pH 7 pyrophos- 

 phate at 30° C, but 18 hours at 25° C. are more generally used. 



Denatured protein precipitates under the influence of 0.1 Jf pyrophos- 

 phate. Thus, any precii^itates appearing in reconstitution mixtures are 

 removed by low-speed centrifugation. The amounts of protein in these pre- 

 cipitates are usually quite low, and with fresh, good protein preparations, 

 negligible. The reconstituted virus can be separated from the reaction mix- 

 ture by ultracentrifugation. The amomit of material which is pelleted can be 

 determined by weight, but is more usually derived from the absorbance of 

 the pellet redispersedin water, using 2.7 as the absorbance at maximum (260- 



