VII. SYNTHESIS OF RXA AXD RIBOSOMES 311 



somes which earn' a particular protein by the antibody to that protein. 

 Ribosomes have been precipitated by specific antibodies (Cowie et al., 

 1961; Warren and Goldthwait, 1961) but analysis shows only a marginal 

 difference in base composition from the average (Midgley and Gold- 

 thwait, 19611. 



J\. Met.4BOLic Pools 



For kinetic studies of ribosome sjTithesis to be interpretable in a 

 quantitative way a number of conditions must be met. Among these, 

 two are of primary' importance. First, the cells must be growing in 

 steady-state conditions so that cellular components are not changing in 

 their proportion. Second, the time course of the specific radioactivity 

 of the immediate precursors for macromolecular synthesis must be 

 known. 



The first condition can be satisfied by allowing a prolonged period 

 of exponential growth (in an adequate and unchanging medium) before 

 the introduction of the tracer. The addition of a tracer such as S^^O^" or 

 P^-Oi — to a medium which already contains S'-Oi" and P'^Oi intro- 

 duces no disturbance. In principle, labeled amino acids or bases should 

 be added to a medium which already contains the unlabeled amino acid 

 or base without changing the concentration. In practice, however, this 

 procedure is wasteful of the tracer, and no transient in the rate of 

 macromolecular synthesis occurs as a result of the change from an 

 endogenous to an exogenous supply of amino acids or bases. The cus- 

 tomary- procedure is therefore to add a labeled amino acid or base to a 

 culture already growing exponentially on its endogenous supply. De- 

 ficient mutants should not be used in this way as the addition of the 

 required (labeled) nutrient to a starved cell would initiate growth, 

 steady-state conditions would not apply, and the results would not be 

 meaningful. 



The second condition requires a technique for the evaluation of the 

 specific radioacti^-ity of the precursors to the macromolecules. In 

 E. coli there exist pools of amino acids in the cell so that exogenous 

 tracer amino acids may be diluted by endogenous amino acids upon 

 entn,-. Also there is exchange between exogenous and endogenous amino 

 acids so that even the external material can be diluted. Following the 

 addition of an amino acid to the medium, it is concentrated (as much as 

 5000 times) in the cells and depleted from the medium. Furthermore, an 

 amino acid may be converted to another before incoi-]X)ration into 

 protein. These reactions would be difficult to follow in detail by any 

 direct method. 



Fortunately, when steady-state conditions prevail, the required 



