INTRACELLULAR MULTIPLICATION OF BACTERIAL VIRUSES 253 



walls. Kellenberger and Kellenberger (1957), finally, made electron optical 

 observations on the production of phage-related structures in single infected 

 bacteria and found that previous conclusions concerning the appearance of 

 doughnuts drawn from study of mass lysates are also valid at the level of the 

 individual cell. Figure 1 shows an electron micrograph in which morpholo- 

 gically intact phages, empty heads, and rods are visible. 



In contrast to the serum-precipitable, sulfurylated, mcomplete phage 

 antigens, the precursor nature of the SBP antigens and of the empty heads 

 has not yet been clearly established. To be sure, both of these incomplete 

 materials are found within the phage-infected cell prior to the appearance of 

 any infective progeny particles, but it is difiicult to demonstrate that incom- 

 plete SBP antigens or empty heads present at an early stage of the latent 

 period form part of intact viruses later on. The fact that the intracellular 

 concentration of both of these incomplete materials reaches a maximum soon 

 after the termination of the eclipse has been interpreted in favor of the 

 precursor view by supposing that this constant level reflects a steady state of 

 depletion by conversion into mature progeny and replenishment by renewed 

 synthesis (Levinthal and Fisher, 1952; DeMars, 1955). 



4. Phage-Specijic Nucleic Acid 



Although the experiments discussed so far demonstrate the appearance of 

 noninfective proteinaceous structures already during the eclipse, they fail to 

 reveal any incomplete forms of the phage DNA, the other major component of 

 the mature virus. Fortunately, in the case of the T-even bacteriophages, it is 

 possible to examine infected bacteria for any phage- specific nucleic acid by 

 taking advantage of the discovery that the DNA of these phage strains 

 contains the unusual pyrimidine, 5-hydroxymethylcytosine (HMC), in place 

 of the more common base, cytosine, of the DNA of the E. coli host bacterium 

 (Wyatt and Cohen, 1952). Hershey et al. (1953), therefore, infected E. coli 

 bacteria under the conditions of the one-step growth experiment, extracted 

 the DNA of the infected complexes at various times after the onset of phage 

 development and analyzed the extract for its content of HMC, cytosine, and 

 the other purine and pyrimidine bases. These analyses then permitted an 

 estimation of the number of phage equivalents of HMC-containing DNA 

 present at any time, based on the total nucleic acid and relative HMC content 

 of the mature phage particle. Vidaver and Kozloff (1957) later repeated the 

 study of Hershey et al., paying, however, closer attention to the very earliest 

 stages of the latent period in order to establish more precisely the onset of 

 synthesis of HMC-containing DNA. The results of these experiments can be 

 summarized by stating that the synthesis of bacteriophage DNA already 

 commences between 6 or 7 minutes after infection and then proceeds so 

 rapidly that a few minutes later at the termination of the echpse, when the 



