KOHNE: In histological studies, from what 

 I've seen, the cells look about the same size. 



KAHN: Does developmental arrest of the 

 catesbeiana-pipiens hybrids occur at the same 

 stage in reciprocal crosses? 



KOHNE: No, it's different. An interesting 

 thing about hybrids in general is that they 

 react differently depending on which egg cyto- 

 plasm is used. Even though the same total 

 genome is present in the different cytoplasms 

 the end result may be quite different. 



EPEL: Are the magnesium-deficient and 

 the anucleolate embryos the same? 



KOHNE: No, but they are almost pheno- 

 copies. They act more or less the same way, 

 except the magnesium-deficients obviously do 

 synthesize ribosomal RNA. 



POLLARD: Do you have any kind of hy- 

 pothesis? For example, can we say the following? 

 The idea is that one chromosome or one part 

 of a chromosome has the mechanism for tran- 

 scribing the ribosomal RNA. This has to come 

 off the DNA. The DNA you have is no good, it 

 won't work. It'll transcribe all right in one, but 

 it won't in the other, so it's stuck with the 

 wrong transcription. It keeps pumping this out 

 and this hooks up with the RNA and it doesn't 

 work. The stage in which you need this ribosomal 

 RNA could come quite a bit earlier than when 

 the cell is desperate for it. 



KOHNE: Yes, I agree with you. 



POLLARD: The concentrations may be 

 very critical. You may need just 8 or 10 ribos- 

 omes to get something started. It seems to me 

 you've got to have the organism tell you when 

 you're supplying a deficiency. I would start 

 with a nice arrested cell with everything in bad 

 shape and then start firing things into it. I 

 would use anything I could think of that was 

 remotely similar to transcriptase, anything 

 I could get off a DNA, and any kind of histone 

 material which was somehow associated with 

 DNA. I'd try to get hold of something that would 

 unlock this mechanism. What's the matter with 

 that idea? 



KOHNE: Well, I'm overwhelmed. 



GROSS: Dr. Pollard, why do you fire in 

 transcriptase? 



POLLARD: Well, it seems to have every- 

 thing else. 



GROSS: It's also got transcriptase. 



POLLARD: Yes, but what it has won't work. 



GROSS: I think I'd try naked ribosomes. 



POLLARD: Would you use ribosomes made 

 on the DNA in the hybrid? 



KOHNE: The haploid is perfectly capable 

 of making ribosomes; the only thing the haploid 



doesn't have that this hybrid has is the other 

 set of chromosomes. 



POLLARD: Maybe you've got the right 

 transcriptase and the wrong DNA. 



KOHNE: Well, each of these things con- 

 tributes a nucleolus. I think that the cytoplasm 

 interaction with this cafes ftezana genome is what 

 stops development. However, what it is really 

 doing I don't know. 



PAPACONSTANTINOU: If the nucleoli don't 

 appear when they should appear, right before 

 gastrulation, I don't understand why you think it 

 has to be cytoplasmic. Do you think something 

 in the cytoplasm is regulating the appearance 

 of the nucleolus? 



KOHNE: Yes, let me talk about that in a 

 minute. This is all really pretty much specula- 

 tion. We don't have many facts to go on. 



TS'O: Have any practical chemical tests 

 been done here? 



KOHNE: I don't think anybody's ever done 

 any on catesbeiana. That's something I've al- 

 ways wondered about. What differences are 

 there qualitatively and quantitatively in the 

 DNA's of these frogs. Amphibians have a lot of 

 DNA in their cells compared to other things. 



GROSS: Let us go back to some of your 

 other points. Is there a fixed quantitative rela- 

 tionship between cytoplasm, DNA and RNA? 

 Maybe there is a cytoplasmic repressor. 



KOHNE: Possibly. I'm just saying that cy- 

 toplasm is somehow involved in control of 

 nucleic acid synthesis. These cells also divide 

 extremely rapidly during development. 



POLLARD: You're saying that up to tetra- 

 ploid in your system, the relationship of cyto- 

 plasmic volume to RNA and DNA seems to be 

 very constant? 



KOHNE: I was quoting other people's work. 

 In 1925 de Beer quoted experiments related to 

 cytoplasmic-nuclear ratio. He suggested that 

 what controls the development of embryos is 

 the cytoplasmic-nuclear ratio. He stated that 

 when you get to a certain stage the genome is 

 turned on and that what triggers it is the ratio 

 of the nuclear material to the cytoplasmic 

 material. He pinpointed that stage at gastrula, 

 the same stage at which we know now that 

 messenger RNA synthesis is turned on very 

 rapidly. I have changed the wording a little to 

 the parlance of molecular biology in saying that 

 the cytoplasm is controlling the extent of DNA 

 synthesis and that a DNA-cytoplasm interaction 

 controls RNA synthesis. 



DEERING: Does this 6-day haploid have 

 twice as many cells as the 5-day control? 



KOHNE: That's right. 



44 



