ROUND TABLE DISCUSSION Ti) 
question, because the complicated results influence the interpretation of our experiments on the 
labeling of RNA. When a small quantity of /C-uracil (ro~*? M) is added to a culture of growing 
cells, it is completely taken up within 40 sec (Fig. 5). The radioactivity rapidly enters RNA with a 
delay of less than 5 sec and continues nearly linearly for 40 sec. At this time about 40% of the 
added uracil has been incorporated into RNA. Just when the external uracil is exhausted the rate 
of incorporation into RNA drops abruptly. The curve for the incorporation into RNA after this 
time corresponds to an exponential decay of the radioactivity in a 10-minute pool of uracil com- 
pounds. 
In a comparable experiment done at a very high concentration of uracil (10~* M) the pool was 
relatively so small that it could not be measured accurately. The incorporation of the radio- 
activity into RNA starts again without delay, and slowly accelerates to about twice the initial 
rate with a time constant of about 10 min. These observations as well as those made at a variety 
of intermediate concentration may be interpreted by means of the following diagram. 
Internal synthesis 
External uracil 

+ Incorporation into RNA 
> JE 
t 
5 
P represents a small pool or sequence of reaction steps leading from uracil to a chemical form 
suitable for incorporation into RNA. The quantity in P corresponds to the requirement for about 
5 sec of RNA synthesis. S represents a large pool of compounds which exchange with some uracil 
compound in P. These compounds are principally UDP and UTP, and there is conversion to 
CDP and CTP. The quantity in S corresponds to the requirement for 5 to 10 min of RNA synthesis. 
The rate of exchange between S and P is not fast and equilibrium between the specific radio- 
activity of P and S requires several minutes at least. P then effectively forms a by-pass around the 
large pool, S, for the entry of external uracil into RNA. 
The rate of uptake of uracil into the cell has a maximum value corresponding roughly to the 
rate of utilization of uracil compounds for RNA, and it turns out that these pools are not expand- 
able. The cell very avidly concentrates uracil, with a Kg of 1o~7 molar, but in fact does not increase 
its internal concentration of uracil compounds. Here you have a concentrating process without 
increase in internal concentration. 
Fig. 6 shows the results of an experiment in which the cells were pretreated with 4 Ome Vi 
12C-uracil for 10 min before the C-uracil was added. An identical curve within small limits of 
error was obtained in a simultaneous control experiment in which the !C-uracil and the !C-uracil 
were both added at zero time. The fact that there is still a direct entry of C-uracil into RNA is 
shown in the upper set of curves which are simply a magnification of the early time region. If the 
pool P were expanded there should be an initial delay, which is not observed. The expansion of S, 
on the other hand, would cause a major difference between the control and the experiment in 
which !2C-uracil was added beforehand. The increasing rate of entry of radioactivity into RNA 
shown in Fig. 6 is due to the increasing specific radioactivity of S. If the pool S were expanded, its 
specific radioactivity should rise more rapidly in the control since the new material flowing in 
would be radioactive. On the other hand in the experiment in which !C-uracil was added before- 
hand, the specific radioactivity should rise more slowly since a large amount of unlabeled com- 
pounds would have been present at the time the C-uracil was added. 
I think the argument is fairly rigid that compounds in P must be on special sites, whose proper- 
ties are comparable to my carrier. In the first place, something limits the maximum rate of entry 
of uracil from outside, which cannot go in at a rate faster than it is used, so there is a very ade- 
quate feedback control, on the internal concentration. At the same time there is a feedback control 
on the internal synthesis of uracil. When the concentration is approached where the entry rate 
meets the requirement for RNA synthesis, then the internal synthesis is shut down. 
The mechanisms involved in the binding of the compounds in P are also capable of carrying out 
the conversion of the uracil through all of the stages to whatever compound is required for in- 
corporation into RNA, and including immediate delivery to the sites of RNA synthesis. Thus P 
represents four processes, which are intimately related with each other, and which can by no 
stretch of the imagination be considered to occur at the same location in the cell. They are internal 
synthesis, concentration of external uracil, chemical conversion and transfer to sites for incorpora- 
tion into RNA. 
I think we can presume rather definitely, that the very small quantity of compounds in P is 
associated with some sort of site which I would like to call a carrier. It is rigidly controlled in 
amount as is the large pool with which it is in equilibrium. 
The stability of the large pool, S, leads me to believe that S also is bound to sites. This is sup- 
ported by other experimental evidence, but I don’t think that kinetic arguments could establish 
References p. 777 
