formation of a cap could be a way of protecting 

 the ribosomes from degradation during a time 

 when no synthesis is actually occurring. It also 

 could be an unusual mechanism for controlling 

 protein synthesis by isolating the ribosomes 

 from any one of the many factors involved in 

 the complete functional synthetic system, such 

 as ATP from the mitochondria, for example. 

 It could equally well serve some combination 

 of these. I am stressing these points because I 

 think they provide us with some ideas that can 

 be tested. 



For example, if the cap merely conserves 

 the ribonucleoprotein for synthesis of new 

 ribosomes, germination, then, it ought to be 

 associated with or require concomitant ribosome 

 synthesis at an early stage. K, on the other 

 hand, the ribosomes are conserved as functional 

 units and are actually used as ribosomes with- 

 out alteration, then germination and early pro- 

 tein synthesis might be quite independent of 

 ribosomal RNA synthesis but could require 

 "messenger" RNA synthesis or coding of some 

 kind. Alternatively, if the ribosomes are func- 

 tional, the spore could already be precoded and 

 ready to go; in fact the ribosomes could have 

 the information stored away with them in such 

 a manner that only release from the cap would 

 be necessary. In this case, germination might 

 be completely independent of early RNA syn- 

 thesis of any kind, 



I think we can test these hypotheses. We 

 can isolate the caps and look at them in a cell- 

 free system, for example, to estimate their 

 functional capacity in vitro. We have been trying 

 to get a reliable cell-free system to do this, 

 but we have not yet been successful. 



I would now like to talk about differentiation 

 in terms of the source of the cap ribosomes 

 and then briefly discuss the process of germina- 

 tion, which presents additional clues concerning 

 the particular problem of cap function. 



CHALKLEY: Have you looked at these under 

 the electron microscope to see if there are 

 polysomes there? 



LOVETT: We haven't looked at them in the 

 electron microscope, but we have tried a few 

 inconclusive experiments by isolating the caps, 

 lysing them very gently with detergents, and then 

 layering them on gradients to look for poly- 

 somes. This should show us if there are lots of 

 them. So far, we don't find any. However, we 

 haven't done enough of this to be sure. 



I want to turn to the formation of zoospores, 

 and RNA synthesis in particular, although I'll 

 mention a few other things. First, what happens 



during the differentiation to form zoospores? 

 Figure 7 is a summary diagrm illustrating a part 

 of the life cycle starting with the tiny spore and 

 extending through the exponential growth phase to 

 the mature plant containing many nuclei. Under 

 our conditions, the number of nuclei turns out to 

 be very close to 256, which is somewhat dif- 

 ferent from plants grown by Dr. Cantino's 

 method. Formation of the papillae and subsequent 

 events lead to the formation of the zoospores. 

 This graph is a plotof per cent papilla formation 

 versus culture age to show how sharply the 

 transition occurs in our system. We grow the 

 cultures by inoculating zoospores into a rich 

 medium and then aerating and stirring at 

 24°C (4). At 15/2 hr. we induce differentiation by 

 changing the medium; we don't wait for it to occur 

 normally, although it will do so without induction. 

 However, we get much better synchrony by 

 changing the medium to induce differentiation. 

 This is represented by the fact that the entire 

 population enters this papilla stage, the first 

 obvious morphological event, within a span of 1 

 hr, and 60% of the plants form papillae within a 

 24 min interval. This is pretty good synchrony 

 for an organism with a life cycle of approximately 

 24 hr. 



DEERING: How do you induce this? 



LOVETT: We just change the medium. We 

 wash out the growth medium and resuspend the 

 plants in a dilute inorganic salt solution, the 

 1/2DS you will see indicated in some of the 

 figures. Figure 8 summarizes some of the data 

 obtained for the total protein, RNA and DNA of 

 cultures. You can see that until 15/2 hr, when 

 the medium was changed, all these increased 

 exponentially, but shortly after the change they 

 began to level off: DNA and RNA at about 16,'4 

 hr and protein and dry weight at about 17 or 

 ITA hr. 



Figure 9 illustrates a little more dra- 

 matically, in a nonlogarithmic plot, the pattern 

 observed for whole- cell RNA. You will notice 

 that total RNA continues to increase for a while 

 after the inducation, but then begins to go down- 

 hill, and does so continuously until the end of 

 the cycle at 19'^ hr when the spores are dis- 

 charged. This finally represents a 35% loss in 

 the total RNA of the plant. 



We were interested in these RNA changes, 

 and Figure 10 shows that not only does RNA 

 start to disappear after I6/2 hr, but, if one 

 measures short-term pulse- labeling with C^"*- 

 uracil, the rate of incorporation also drops very 

 drastically between 16 and 17 hr. I want to 

 emphasize the morphological point: the heavy 



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