278 ELLIOT VOLKIN 



iiiiliu'tion iiu'cliaiiisiii llial siiuilaily pioposcs an unstable ciizynic- 

 forming unit that must be synthesized continuously. 



Further experiments with inducible enzyme systems utilizing purine 

 and jiyrimidine analogs not oidy confirm the functional instability of 

 the gene messengei- but suggest that tliis intermediate is indeed a polyri- 

 bonucleotide. The presence of o-fluorouracil results in an alteration of the 

 ^-galactosidase protein whereby its enzymatic activity is drastically 

 reduced, but its antigenic capacity is retained (Bussard et al., 1960). 

 The same analog, 5-fluorouracil, causes the formation of induced alkaline 

 phosphatase jM'otein whose rate of thermal inactivation is greatly in- 

 creased (Naono and Gros, 1960). The important features of the analog 

 effect is the fact that it occurs veiy quickly after administration and 

 thereafter is reflected by a constant proi)ortion (rather than increasing) 

 of abnormal to normal enzyme molecules. The responsible agent, as- 

 sumed to be RNA altered by incorporation of o-fluorouracil into its 

 structure, must be short-lived and not accumulative to account for the 

 observed kinetics. 



B. B.\CTERIOPHAGE INFECTION 



The original experiments (Volkin and Astrachan, 1957) with virulent 

 phage-infected bacteria directly determined that the phage DNA-like 

 RXA undergoes continual turnover. However, they could not distinguish 

 whether such turnover is an obligatory part of its biologic mechanism or 

 whether the turnover is simply a consequence of its catabolism, insofar 

 as no ribosomal or stable RNA accumulates in this system. Further 

 insight was gained by the discoveiy that some DNA-like RNA was 

 synthesized but did not undergo turnover when protein synthesis was 

 inhibited [chloramphenicol inhibition (Astrachan and Volkin, 1959) or 

 deprivation of a required amino acid (Volkin, unpublished observa- 

 tions)]. This technique with T2 infection was more fully exploited 

 (Volkin, 1960) by using as host the mutant B94 (Gollub and Gots, 1959) 

 which requires both adenine and arginine. A prior incubation of T2- 

 infectcd B94 cells in basal medium containing only adenine permits a 

 definite yield of phage upon removal of the adenine and replacement 

 with arginine alone or arginine plus deoxyadenosine (neither condition 

 by itself will allow production of phage). During the preincubation in 

 adenine alone, though essentially no DNA or protein is synthesized, 

 RNA of T2 DNA-like composition steadily accumulates for about 60 

 to 90 minutes. Furthermore, the amount of newly formed RNA (and 

 not any other adenine-containing cell component) can be quantitatively 

 normalized with the subsequent phage yield. The production of T2 

 during the secondaiy incubation is limited, stopping after about 30 



