210 ROBERT L. SINSHEIMER 



or by the use of mutant bacteria specifically requiring certain amino acids, 108 

 it has been shown that protein synthesis in the first few minutes of infection 

 is necessary for the initiation of phage DNA synthesis, but that continued 

 protein synthesis is not necessary for continued DNA synthesis. It has 

 also been shown that protein synthesis is a necessary prerequisite to multi- 

 plicity reactivation." 



(3) Experiments upon DNA Made in the Presence of Chloramphenicol. 

 Thus, by adding chloramphenicol at a time just before the first appearance 

 of phage precursor protein (about 8 to 10 minutes after injection), the DNA 

 pool can be caused to accumulate so that in 45 minutes a pool of some 100 

 to 125 units of phage DNA is produced. If, then, chloramphenicol is re- 

 moved, appropriate experiments indicate that all of this DNA is available 

 for withdrawal into mature phage. 106, 107 Experiments upon the appearance 

 in mature phage of P 32 which had been made available to the infected cells 

 for limited periods either during or after the exposure to chloramphenicol 

 indicate that the DNA made during the presence of chloramphenicol is nor- 

 mal and essentially complete, in that it forms a unitary pool with the pre- 

 sumably normal DNA made after the removal of chloramphenicol. 108 



The chloramphenicol experiments provide a means to accumulate phage 

 DNA which can then be treated in various ways before it is permitted to 

 enter mature phage particles. In this way information is gained about the 

 status and properties of the newly synthesized phage DNA in the cell as 

 compared to the properties of phage DNA when in mature particles. 



Thus, Tomizawa 109 has shown that when ultraviolet irradiation (doses equivalent 

 to 4 or 11 hits to free phage were used) is applied to phage infected cells which have 

 accumulated DNA in the presence of chloramphenicol, and the chloramphenicol is 

 then removed, many of the final progeny phages are noninfective. These noninfective 

 particles contain ultraviolet-damaged DNA. After 11 hits, they are subject to multi- 

 plicity reactivation, photoreactivation, and cross-reactivation to quantitatively just 

 the same extent as phage that have been irradiated as free particles with the same 

 ultraviolet dose. 



If the ultraviolet dose to the cell has not been too heavy (4 hits), DNA synthesis 

 continues after irradiation with little decrease in rate. After a greater dose (11 hits) 

 DNA synthesis is totally blocked although mature phage can be produced after re- 

 moval of chloramphenicol. The number of noninfective particles in the final progeny 

 appears to be directly related to the amount of DNA present at the time of irradiation 

 and is not influenced significantly by the amount of DNA synthesized after irradia- 

 tion. Thus, it appears that the ultraviolet-damaged DNA is able, upon replication, 

 to give rise to normal DNA and that its lesions are not repaired in the process. 



As discussed previously, these experiments performed by adding P 32 

 after irradiation indicate that individual phage particles contain both DNA 



108 K. Burton, Biochem. J. 61, 473 (1955). 



109 J. Tomizawa, Virology 6, 55 (1958). 



