cap ribosomes came from. If a great deal of 

 turnover was going on, it could mean that the 

 ribosomes were actually being degraded and re- 

 synthesized and, in this way, being redistributed 

 from one part of the cell to another. 



The results of a pulse-chase experiment at 



16 hr are given in Fig. 11 and show that we 

 could do labeling and density gradient experi- 

 ments to study RNA synthesis. For this experi- 

 ment the plants were pulsed with C^"* -uracil 

 for 2 min and then "chased" with an excess of 

 "cold" uracil. Zero min indicates the RNA at 

 the start of the chase. The other graphs show 

 the pattern of labeling during the "chase." The 

 first surprising thing we noticed was that 

 these so-called plants have a very animal- like 

 characteristic; they produce a rapidly labeled, 

 heavy RNA that we think, on somewhat indirect 

 evidence, is actually a precursor of ribosomes. 

 It labels rapidly and disappears during the 

 chase as the radioactivity increases under the 

 ribosomal peaks. Actually, we suggest this on 

 the basis of comparison with work on animal 

 tissues where other people have drawn similar 

 conclusions (5). We haven't, however, identified 

 it or proven it to be a ribosomal precursor. 

 To do this it will be necessary to isolate it 

 and do much more work with it. This experiment 

 was done at 16 hr, just before all the interesting 

 events occur. 



Figure 12 illustrates the pattern of RNA 

 synthesis as a function of time. Sixteen hr, 40 

 min is just before synthesis is apparently shut 

 down. The experiment is similar to that on the 

 last slide, except that in this case we only 

 exposed the plants to C^^-uracilfor 10 min, then 

 killed them and extracted the RNA for gradient 

 centrifugation. I only want to point out that this 

 is a typical pattern for transfer-, ribosomal-, 

 and heavy- RNA incorporation, but that these 

 steadily decrease starting at 16 hr until, by 



17 hr, you can't detect any label whatsoever in 

 the RNA fractions. The heavy RNA peak seems 

 to be the last to disappear. 



TS'O: What is the ribonuclease treatment? 



LOVETT: The curve for ribonuclease- 

 treated RNA just shows that if you treat the 

 RNA's in any of these stages, of which we've 

 only shown one, with ribonuclease, that all your 

 material ends up at the top of the gradient. You 

 don't have any in the region of the O.D. peaks, 

 as in the untreated samples. 



This seemed nicely consistent with our 

 earlier results on total RNA and post- labeling, 

 etc., but when we started looking at the pools 

 in terms of Barbara Wright's work (6), which 



HOURS 



Fig. 13. 

 Whole cell uptake vs incorporation Into RNA. 



we needed to do, we suddenly discovered that 

 total uptake versus the amount incorporated into 

 RNA changed drastically during the same period. 

 In other words, there was about a 13-fold de- 

 crease in the ability of uracil to enter the pools 

 between slightly after 16 hr and 17 hr. This 

 meant that we had to re- evaluate our previous 

 interpretation of the labeling patterns as indi- 

 cating a shut-down in RNA synthesis. Perhaps 

 what was being changed was our ability to 

 measure incorporation into RNA rather than the 

 synthesis of RNA itself. 



In order to try to get around this, or at 

 least get some idea of its significance, we did 

 an experiment where we added excess label to 

 a differentiating culture just before 16 hr. The 

 first points were taken right after 16 hr when 

 we knew we could get good uracil labeling in 

 the RNA (Fig. 13). We then followed the dis- 

 tribution of the label as the culture went through 

 the period when we no longer could get at it by 

 pulsing precursors from the outside; thus, we 

 pre-loaded the pools and then followed what 

 happened afterwards. As you can see, the pools 

 were labeled very rapidly. The value for the 

 pool represents the difference between the total 

 label and that in RNA. The counts in the pool 

 increase rapidly, but you will notice that they 

 go up until 17 hr and then the pool size drops 



171 



