376 F. W. STAHL 



about 6 minutes after infection (see Chapter VII, Volume II), (2) This DNA 

 synthesis must be preceded by protein synthesis (see Chapter VII, Volume II). 

 (3) The function of the rll cistrons becomes insensitive to suicide during the 

 first 6 minutes following infection (Steinberg, personal communication). This 

 point \\^11 be expanded in Section III. (4) Recombination is stimulated by 

 irradiation of stable (10-minute) complexes. This point is discussed below. 



2. Effects of Ultraviolet Light on Vegetative Phage 



Tomizawa (1958) irradiated infected cells after the attainment of resist- 

 ance and then examined the emerging progeny phage. At sufficiently high 

 dose, all the emerging progeny were unable to form plaques. In multiplicity, 

 cross, and photoreactivation experiments they showed the response char- 

 acteristic of a population of phages which had been irradiated in the free 

 state with the same dose of ultraviolet light. 



Hershey (1958) irradiated cells infected with two genotypes of phage after 

 the onset of DNA production. The fraction of recombinants in the progeny 

 increases with increasing dose. Furthermore, the yields from such cells at 

 high doses tend to be genotypically pure (Hershey, personal communication). 

 As we have seen, this is exactly the genetic consequence of multiplicity 

 reactivation when the irradiation is given prior to uifection (Epstein, 1958). 



III. The Inactivation of T-Even Phage by the Decay of 

 Incorporated P^^ (Suicide) 



A. Basic Procedures 



1. Preparation of Radioactive Phage 



Phage containing large amounts of P^^ are prepared by infecting with a 

 small number of phage particles a culture of cells which has been growing in 

 medium of high specific activity. The inoculum of phage is usually so ad- 

 justed that after two cycles of infection the great majority of the cells have 

 lysed. The phage are then freed if necessary from the radioactive culture 

 medium and cell debris and stored at sufficiently low concentration that the 

 beta ray activity has no significant efi*ect on the viability of the particles. 



2. Survival Curves 



The P32 within the DNA of the phage particles decays according to the 

 equation 



N = Noe-^ (10) 



where A is the decay constant of the isotope, Ng is the original number of 

 P32 atoms, and N is the number remaining at time t. For t measured in days, 

 A is about 0.05. 



