DEERING: Are the cells smaller? 



KOHNE: Yes, they are. Again this is a gen- 

 eralization because I can't say anything about 

 what happens to the cells of specific parts, 

 such as those destined to be the liver, brain 

 or epidermis. 



GRUN: There is one thing I was wondering 

 about. This is the comparison between haploids 

 and diploids. I wonder, has any attempt been 

 made to produce inbred lines of frogs? What 

 I'm concerned with is the question of whether 

 this is a straight line comparison of the haploid 

 state as compared with the diploid state or 

 whether you're saying that the condition is a 

 genetic effect of exposed recessives that, of 

 course, would be effective in the haploid state 

 and cause abnormal development. 



KOHNE: Well, as I mentioned, if you start 

 with a small enough egg, you will get normal 

 development, so the phenomenon is probably 

 not caused by the expression of recessives. 



KAHN: Have you considered the possible 

 role of cytoplasmic DNA? 



KOHNE: There hasn't been any evidence 

 as yet that cytoplasmic DNA is in any way active. 

 Igor Dawid at Carnegie Institution has isolated 

 a substance that has the characteristics of non- 

 nuclear DNA, and he thinks it may come from 

 mitochondria. Whether it's active in the differ- 

 entiation process I don't know. 



KAHN: Isn't it true that DNA synthesis does 

 not begin until the beginning of gastrulation? 



KOHNE: No, it begins immediately. Many 

 people have thought that the cytoplasmic DNA 

 might be contributing to the genome, but it's 

 never been proven. There are several systems 

 now in which immediate DNA synthesis has 

 been shown. 



GROSS: It doesn't make any difference 

 quantitatively - the new DNA in the amphibian 

 doesn't begin to make an impact on the total 

 DNA per egg for some time. 



KOHNE: There is supposed to be about 1000 



times more cytoplasmic DNA than nuclear DNA 

 in Rana eggs and 300 times in sea urchin. 



GROSS: At any rate, there is a lot of it 

 around, and even if each genome is fully repli- 

 cated from the pool, the impact on the total 

 DNA will be small until you get about a thousand 

 cells or so. His ratios are all taken at stages 

 where, presumably, the cytoplasmic DNA has 

 been used up. 



KAHN: This raises other questions. How 

 is the cytoplasmic DNA utilized? What is the 

 function of mitochondrial DNA? 



KOHNE: There are two sources of diphen- 

 ylamine-reacting material (DNA like). One of 

 them is the mitochondrial DNA, The other one 

 is an acid soluble fraction and is probably 

 just nucleotides. There is about 10 times as 

 much of the latter as there is of the DNA poly- 

 mer. 



GROSS: It may turn out that the thing people 

 have been overlooking systematically is the 

 enormous ratio of cytoplasm to nucleus in the 

 egg. Since the sizes of mitochondria don't 

 differ greatly between embryonic and somatic 

 cells, this may mean that in the egg you have 

 thousands of times as many mitochondria per 

 nucleus as you have in the somatic cell. And if 

 all mitochondria do have DNA, then in the egg 

 the mitochondrial DNAmight make a tremendous 

 impact on the total, whereas in a somatic cell 

 it wouldn't. 



KOHNE: There is some initial circum- 

 stantial evidence, in studies on centrifuged 

 ascidian eggs, that you get two fractions: one 

 of them with mitochondria and the other without. 

 The part with mitochondria will develop and the 

 part without won't. For another type of ascidian 

 with a light mitochondrial fraction and a heavy 

 mitochondiral fraction, you get a partitioning 

 of mitochondria in each fraction and both will 

 develop: one of them being haploid and the 

 other being diploid. However, without the mito- 

 chondria these things don't develop. 



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