44 



VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH 



that shown in Table III, and the content of P^^ was only slightly less after 

 lysis. The phage liberated during freezing and thawing was also titrated in 

 this experiment. The lysis occurred without appreciable liberation of phage 

 in suspensions frozen up to and including the 16th minute, and the 20 min- 

 ute sample yielded only five per bacterium. Another sample of the culture 

 formalinized at 30 minutes, and centrifuged without freezing, contained 66 

 per cent of the P^^ in non-sedimentable form. The yield of extracellular phage 

 at 30 minutes was 108 per bacterium, and the sedimented material consisted 

 largely of formless debris but contained also many apparently intact cell 

 membranes. 



TABLE III 



Sensitization of Intracellular Phage to DNase by Freezing, Thawing, and Fixation 

 with Formaldehyde 



Unadsorbed 

 phage frozen, 

 thawed, fixed 



Infected cells 



frozen, thawed, 



fixed 



Infected cells 

 fixed only 



Low speed sediment fraction 



The figures express per cent of total P'^ in the original phage, or its adsorbed fraction. 



We draw the following conclusions from the experiments in which cells 

 infected with P^"-labeled phage are subjected to freezing and thawing. 



1. Phage DNA becomes sensitive to DNAse after adsorption to bacteria in 

 buffer under conditions in which no known growth process occurs (Benzer, 

 1952; Dulbecco, 1952). 



2. The cell membrane can be made permeable to DNase under conditions 

 that do not permit the escape of either the intracellular P^- or the bulk of the 

 cell contents. 



3. Even if the cells lyse as a result of freezing and thawing, permitting escape 

 of other cell constituents, most of the P^^ derived from phage remains inside 

 the cell membranes, as do the mature phage progeny. 



4. The intracellular P'^ derived from phage is largely freed during spon- 

 taneous lysis accompanied by phage liberation. 



92 



