starts at about 35-40 min and this is the same 

 time that actinomycin really inhibits develop- 

 ment. The cells won't go beyond this stage. 

 However, this doesn't necessarily mean that a 

 lack of ribosomal-RNA synthesis is the problem. 

 It could be that pre-coding was exhausted at that 

 time and the cells would have to make new 

 messenger. We don't have any idea yet. 



PAPACONSTANTINOU: What is the status 

 of the nucleolus at that stage? 



LOVETT: I forgot to mention that. The 

 spores have a very small, compact nucleolus 

 always located at the base of the nucleus oppo- 

 site the flagellum. In plants, the nucleolus may 

 include almost 60% of the nuclear volume. From 

 less than 10% of the volume of the nucleus in a 

 motile spore, it goes to about 50-60% of the 

 nuclear volume in an actively synthesizing 

 germling. With actinomycin treatment, they stay 

 small. However, we have not made any critical 

 measurements and this is a very rough esti- 

 mate. 



PAPACONSTANTINOU: I was just talking 

 about the experiment that came out about a 

 year ago from the Massachusetts General Hos- 

 pital where they isolated the nucleoli from 

 B. emersonii I believe. 



LOVETT: Well, they said they did. They 

 didn't prove it by any manner of means. 



PAPACONSTANTINOU: I was wondering 

 about that. Also, they claimed that they isolated 

 a specific DNA whose base ratio was comple- 

 mentary to the ribosomal-RNA. 



LOVETT: We haven't done anything like 

 this at all. I'm not able to interpret some of 

 Dr. Comb's work because he doesn't describe 

 his methods very completely, and I can't really 

 evaluate it. 



MAURER: Did you try cycloheximide as an 

 inhibitor of protein synthesis? 



LOVETT: No. We did try chloramphenicol 

 and it had no effect, but it doesn't seem to be 

 very effective with fungi in general. 



McCARL: Was that graph you had on the 

 board with the 4S peak the labeling? 



LOVETT: Yes, this was the labeling from 

 uracil during a short incubation of 5 min. 



McCARL: This shows synthesis? 



LOVETT: I don't know specifically. This is 

 the same question that Paul has; we don't know 

 whether it is addition of terminal groups, whether 

 it represents new synthesis, or what. We have 

 to get some of it out and look at it. 



GROSS: Are you worried about end- 

 labeling? 



LOVETT: No, I can get cytosine very 



easily. That's no problem. In fact, Tm sure this 

 is true. 



EPEL: Concerning actinomycin effects, 

 this could be added during the first 30 min of 

 germination with no effect? 



LOVETT: We can let them germinate in it. 



EPEL: Have you pulsed actinomycin? 



LOVETT: No, we haven't tried removing it. 

 We've tried it where they germinate in it and 

 we've tested it where we take them out of a 

 normal culture as a function of time and put 

 them in it. You get the same results either 

 way; that is, actinomycin doesn't have any 

 effect until a certain point and then they be- 

 come sensitive. 



EPEL: If you add actinomycin during spore 

 formation they still form spores, but are these 

 spores then capable of germinating? 



LOVETT: Yes, they seem to be. We haven't 

 really tested this by growing them, but they seem 

 to be perfectly normal spores. I think that this 

 means that all the important events have hap- 

 pended by that time. It is interesting that the 

 "packaging" of the ribosomes is one of the very 

 last events. It does make sense that they are 

 used for protein synthesis and then packaged 

 up when practically everything essential is 

 done. This occurs at about 10 min before 19 

 hr, and it is very soon after 19 hr that the 

 spores are actually released. 



GRUN: There were some small particles 

 in your electron micrographs that resembled 

 amyloplasts in a vague sort of way. Do you know 

 what I mean? 



LOVETT: Are you referring to the little 

 round ones? 



GRUN: There were some little round things 

 with a darker, fairly homogenous stain. 



LOVETT: There are some little cup- shaped 

 structures in vesicles that are present in ap- 

 proximately the right numbers to be the particles 

 that stain with the Nadi reagent, as Ed reported 

 some years ago. These apparently occur only 

 in the spore, since we have seen them nowhere 

 else. I have no idea as to their function. 



CANTINO: Their number depends upon the 

 kind of plant which formed the spores and it can 

 be modified by environmental changes, (cf. 

 F. C. Cantino and E. A. Horenstein, Mycologia 

 48, 443, 1956). 



GRUN: They don't contain starch, do they? 



LOVETT: I doubt it. I don't know, to be 

 quite honest with you. 



GRUN: They were near the membranes in 

 the electron micrograph. 



LOVETT: They're very characteristic look- 



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