the same. I think, however, that the point I am 

 trying to make with respect to the regulatory 

 mechanism has nothing to do with the genes; 

 it's at an entirely different level. The only 

 genetic involvement in this work is the regula- 

 tion of subunit synthesis. 



MASSARO: On the basis of the pyruvate 

 inhibition curve alone, I don't think you can 

 emphasize that these LDH's are one and the 

 same. 



GROSS: It seems to me if you have two 

 different genes, then the original explanation is 

 wrong on the basis of Papaconstantinou's re- 

 sults. If you don't have two genes, then this 

 is another complicated case of differential gene 

 action and not very much more can be said 

 about it. 



HYMER: Have you studied the process of 

 ribosomal breakdown during differentiation to 

 the fiber cell by electron microscopy? 



PAPACONSTANTINOU: With electron mi- 

 croscopy there is a decrease in the ribosomal 

 population. In other words, if you count the 

 ribosomes in a unit area, you'd find a decrease 

 in the actual number of ribosomes. 



HYMER: Are these membrane bound? 



PAPACONSTANTINOU: No, they're not 

 membrane bound, especially in the fiber cells. 



KOHNE: The one criticism that you could 

 make of this is that RNA breakdown is some- 

 thing other than ribosomal breakdown since all 

 you've got is phenolyzed RNA. 



PAPACONSTANTINOU: Well, there is no 

 ribonuclease in the lens system, that we can 

 detect. I apologize to everybody who has ribo- 

 nuclease troubles, but we just don't have them. 

 We used 5 gammas/ml of polyvinylsulfate in 

 our phenol extractions but it doesn't make any 

 difference whether we use it or not. 



POLLARD: How does ribosomal RNA break 

 down? 



PAPACONSTANTINOU: We don't knowyet. 

 It's been a very interesting phenomenon. If 

 ribonuclease is present we ought to get break- 

 down products smaller than what we observe, 

 unless it's a special ribonuclease, which I'd 

 like very much to find out. 



GROSS: You remember Nemer's paper on 

 RNA synthesis during the early development of 

 the sea urchin, in which he showed gradients 

 with RNA's that looked like 28s, 18s, 13s, 10-13s 

 and 4s? The 28s RNA in animal cells of this 

 sort does, in fact, break down into things that 

 sediment roughly at 10- 13s. 



PAPACONSTANTINOU: Because of the high 

 GC content of the breakdown product I am in- 



clined to believe that it comes from the 50s 

 ribosomal particle. This is the part of the ribo- 

 some that 28s RNA is derived from. However, 

 this is just speculation right now. We are trying 

 to characterize the 50s and 30s ribosomal RNA 

 to determine if the breakdown product originates 

 specifically from either one or both. 



EPEL: Can you say a little more about the 

 temporal relationship between the ribosomal 

 breakdown and the lens protein synthesis? 



PAPACONSTANTINOU: Although we don't 

 really have kinetics or good turnover data, 

 indications are that lens protein synthesis 

 decreases about 10-fold in going from an 

 epithelial cell to a fiber cell. Paul, did you 

 mention the fact that there was a protein that 

 was involved in the stabilization of messenger 

 RNA in the early embryo? 



GROSS: It's an idea that's been suggested. 

 Monroy and his colleagues found that trypsin 

 treatment of unfertilized ribosomes allowed 

 them to work. 



PAPACONSTANTINOU : If we prepare ribo- 

 somes from epithelial cells and fiber cells and 

 do an RNA-protein ratio, the RNA-protein ratio 

 in the epithelial cells is about 0.5 to 0.8 (they're 

 good ribosomes). When you do it in the fiber 

 cell, it goes down to 0.1 and sometimes less. 

 There' s an indication that there' s protein being 

 stuck onto the ribosomes of the fiber cells 

 which may explain that smooth endoplasmic 

 reticulum and possibly the stabilization of 

 messenger. 



GROSS: Couldn't there also be isolation 

 artifacts since you're making lots of the crys- 

 tallins and what not? 



PAPACONSTANTINOU: Well, no. We're 

 extracting under the same conditions. Why 

 shouldn't we get a lot of protein on these ribo- 

 somes then? 



GROSS: Because it's a different kind of 

 protein. 



PAPACONSTANTINOU: The only differ- 

 ence we have is the formation of the gamma 

 crystallin. The gamma crystallin is a basic 

 protein relative to the others. The gamma 

 crystallins could be sticking onto those ribo- 

 somes. They may be a stabilizing factor. This 

 is a highly specific protein that is formed in 

 fiber cell formation. This is a real speculation, 

 though. It's a highly specific protein that's 

 associated mainly" with fiber cells and it comes 

 up just before the stabilization of the messenger. 

 I don't know why they would want so much 

 gamma crystallin for the stabilization. 



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