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plished by means of a uracilless mutant which was forced to synthesize proteins 

 rapidly. It was found that the free nucleotide pool was considerably depressed 

 in a matter of 30 minutes. It was possible to replenish this pool rapidly by in- 

 cubation in the presence of mixtures of purine and pyrimidine bases in the prop- 

 er ratio. Restoration of enzyme-forming capacity in cells whose nucleotide 

 pool had been depleted could be obtained very quickly by incubation with the 

 proper ratio of purine and pyrimidine bases. 



CHARGAFF: What is the right ratio? 



SPIEGELMAN: The ratio we employed corresponded to the published 

 analysis of the RNA of yeast. These data argue against nonspecific effects 

 which might be attributable to the polyphosphate of the purines and pyrimidines 

 in energy transfers and other reactions. If one takes a cell that is half induced, 

 and, therefore, has the right systems to form the particular enzyme being 

 studied, and depletes the nucleotide pool, any further capacity to form more en- 

 zyme molecules is abolished. The data thus far, would seem to indicate that 

 the cell must be able to make new RNA if it is going to synthesize new enzyme 

 molecules. It does not appear to be necessary for a cell to synthesize new DNA 

 in the process of forming enzymes. 



MAZIA: I think, that evidence for the nuclear origin of RNA, which 

 has been fairly good, is growing stronger. 



I should like to show you some simple experiments done on the amebae 

 that bear on this. The amebae received no external nutrition during these ex- 

 periments; we are dealing with endogenous processes. 



Brachet had shown, a few years ago, that if you cut an ameba in half 

 and follow the total RNA content of the half with the nucleus and of the half with- 

 out a nucleus (in the absence of external food sources), you obtain the following 

 result: the RNA content of the half with a nucleus declines very slowly over a 

 period of days, while the RNA of the enucleated half declines steadily and rapid- 

 ly. You could interpret these experiments as meaning either that the nucleus 

 was stabilizing the RNA or that it was replacing RNA nearly as fast as it was 

 broken down. 



James, in our laboratory, was doing the same experiments at about the 

 same time, but added a simple control: He followed the RNA content of intact 

 amebae kept under the same conditions. Much to our surprise, the RNA content 

 of these whole amebae dropped with time in a way that resembled the situation 

 in enucleated halves more than that in nucleated halvesl 



At first glance, the experiment would suggest that Brachet was wrong 

 in concluding that the nucleus was involved in maintaining the RNA level in the 

 cell, for these whole amebae also had perfectly good nuclei. But a simple and 

 interesting calculation by James showed that the new data actually proved the 

 existence of a nuclear activity that is responsible for the maintenance of the 

 RNA level of the cytoplasm as postulated by Brachet. Let us consider the sim- 

 plest kind of replacement mechanism; namely, one in which the RNA in the cy- 

 toplasm is continuously breaking down, and can be replaced only by the activity 

 of the nucleus. In the experiments of Brachet and of James, we have the simple 

 condition that nothing is entering the cell from the outside; we are dealing with 

 endogenous processes. Then we may use the rate of decrease of RNA in the 

 enucleated half as d measure of the rate of breakdown of cytoplasmic RNA. The 

 difference between the lower rate of loss of RNA by the nucleated half and that 

 of the enucleated half gives us, by simple arithmetic, the rate of replacement of 



