URACIL-C 



10 20 30 



MINUTES 



Fig. 25. 



Precursor incorporation by zoospores germinating in an 

 organic medium. 



can use to try and interpret, or to fit into the 

 morphological sequence, the points I have just 

 discussed. Zoospore germination begins between 

 12 and 15 min after inoculating the spores into 

 the culture medium (Stage 1). It doesn't occur 

 sooner because the harvested spores are held on 

 ice in very concentrated suspensions while being 

 centrifuged and washed. Our germination experi- 

 ments start when the zoospores are diluted up 

 in the medium at 24°C. Twelve to 15 min later, 

 they roimd up and begin to germinate. The next 

 to last picture in the sequence (Stage 4) is a 2 hr 

 plant with a well- developed rhizoid. 



The previous slide showed that a measur- 

 able increase in RNA and protein cannot be ob- 

 served until 20 and 40 min, respectively. The 

 next figure demonstrates that incorporation of 

 C^^ -uracil or C^'* -leucine can be detected much 

 earlier (Fig. 25). Uracil incorporation occurs by 

 10 min, if not sooner, as does leucine incorpora- 

 tion, which here looks somewhat slower than 

 it actually is because it has not been corrected 

 for its differing specific activity. Such a correc- 

 tion would move it much nearer to the RNA 

 curve. 



While I have no further data to present, I 

 would like to mention a few recent and interest- 

 ing results. First, the pattern of synthesis and 

 early differentiation of the spores - up to a 

 stage where they form a tiny, uninucleate plant 

 with a fairly long, branched rhizoid - will occur 

 whether you put the spores in the growth medium 

 or not. The spore appears able to accomplish 

 this quite well at the expense of whatever it 

 carries with it. The lipid and polysaccharide 



granules may serve as energy supplies for this. 

 The same is true for precursor incorporation. 

 Thus, the early events in germination appear to 

 be nearly independent of the medium. The trig- 

 gering of the spores to germinate may not be 

 independent of the medium, but we do not know 

 what sets the process in motion. 



Now to go back to some of the problems I 

 posed earlier concerning the function of the cap 

 which we have obviously not really answered. As 

 I said before, the reappearance of the ribosomes 

 in the cytoplasm at germination could result 

 from synthesis of new ribosomes at the expense 

 of the cap ribonucleoprotein, or from migration 

 of the pre-existing ribosomes. In the first case, 

 ribosomal RNA synthesis would be required. The 

 second might necessitate coding of the ribosomes 

 for early protein synthesis, or it might require 

 nothing. This is an area of great interest to us, 

 and I would like to mention a few preliminary 

 experiments which are not yet at the stage where 

 I am ready to put them on a slide. 



First of all, zoospore germination will pro- 

 ceed to between stage 3 and stage 4 (Fig. 24), 

 when it has just produced a short rhizoidal out- 

 growth, whether it is in actinomycin D or not. 

 In other words, if one adds 25-100 |/g/ml of 

 actinomycin the spore swims normally, settles 

 down, rounds up, the cap breaks down, and the 

 primary rhizoid is produced. At this point 

 further development is completely inhibited. 

 That is one observation. We know from other 

 experiments that during the same period, 

 25 //g/ml of actinomycin is very effective in 

 inhibiting uracil incorporation. We haven't 

 looked at the RNA yet. 



TS'O: At that stage is there any evidence 

 that actinomycin is actually going in? 



LOVETT: Well, I assume it goes in because 

 it completely inhibits uracil incorporation which 

 occurs at the same stage. 



TS'O: However, can it still get in the later 

 part? 



LOVETT: It completely inhibits the later 

 stage, so I assume it's getting in, though I don't 

 have direct evidence. It is still inhibiting after 

 that time, at least in control experiments, where 

 we have had no actinomycin present earlier; by 

 this I mean that it still inhibits uracil incorpora- 

 tion at the later stages. The third point is that 

 actinomycin seems to have practically no effect 

 on leucine incorporation during the first 30 min; 

 so far, we haven't followed it much beyond that 

 point. We have put the spores in 25 /ig/ml of 

 actinomycin, allowed them to germinate, and 

 measured leucine incorporation, with C^^- 



178 



