INTRACELLULAR MULTIPLICATION OF BACTERIAL VIRUSES 261 



of about 20 phage equivalents per cell. It could be shown, furthermore, in 

 experiments in which the P^^ label was either added to or withdrawn from 

 the culture at various times after infection, that phosphorus atoms con- 

 tinuously enter and leave this postinfection E,NA species, i.e., that this 

 fraction is in a state of rapid metabolic turnover, in contrast to the KNA of 

 normal, rapidly growing E. coli cells for which no such turnover exists 

 (Hershey, 1954). Whatever may be the importance of this postinfection RNA 

 for phage multiplication, it cannot be a direct precursor of the infective 

 progeny, since the extracellular T-even particles contain no RNA (Putnam, 

 1953; Volkin and Astrachan, 1956b). 



B. ''Early" Protein 



We have already noted that, in contrast to the immediate cessation of the 

 net synthesis of RNA, the formation of bacterial protein continues imimpaired 

 after infection of the host cell. Since the synthesis oi phage precursor protein, 

 however, does not commence until the latter part of the eclipse period, it 

 might be thought that the protein synthesized within the infected cell in the 

 time intervening between infection and the onset of precursor manufacture 

 is not directly connected with the process of phage reproduction. The 

 experiments to be recounted now demonstrate that, on the contrary, a 

 protein species arises among these nonprecursor proteins formed immediately 

 after infection, which is an essential factor of intracellular phage development. 

 Cohen and Fowler (1947) found that there is no phage growth if the amino 

 acid analog, 5-methyl-tryptophan, which arrests protein synthesis by block- 

 ing the biosynthesis of tryptophan (Beerstecher, 1954), is added to bacteria at 

 the moment of their infection with T-even phages. From this work it already 

 appeared that the observed interference with phage growth could not be due 

 just to the inhibition of synthesis of the phage protein, but must represent an 

 interference with a very early step in the reproduction process, since net 

 synthesis of DNA by the infected cell also is suppressed. After removal of 

 5-methyl-tryptophan from the infected culture, phage development com- 

 mences, leading to a burst of progeny phages after a normal latent period 

 reckoned from the time of removal of the inhibitor. Subsequent refinements 

 and extensions of these studies showed that there is no synthesis of phage DNA 

 or onset of phage development in infected bacteria in which protein synthesis 

 has boen suppressed from the very start either by the presence of an inhibitory 

 amino acid analog, by the absence of required amino acids, or by the presence 

 of the antibiotic, chloramphenicol. If, however, the bacteria are infected 

 under normal conditions and protein synthesis is stopped by any of these 

 methods only a few minutes after the start of intracellular phage development, 

 then the synthesis of HMC-containing phage DNA does proceed in the 

 absence of any further formation of protein, the rate of such DNA synthesis 



