116 



MAZIA: He has obtained a 19-fold increase in spontaneous chromo- 

 some breaks by raising plants on calcium -deficient media. The Ca-deficiency 

 cannot be too severe, or the plants will not grow at all. Holding the Ca-supply 

 at a marginal level, he still observes the 19-fold effect. 



PLATZMAN: Is it spontaneous? 



MAZIA: Yes. I don't know whether there are any data on radiation 

 effects yet. 



CURTIS: Not yet. 



POLLARD: If you measure the dependence of this effect, then you know 

 what the bond is. If you can show that this is spontaneous, at a rate which is 

 not very high, you have an ideal bond. It overlaps. 



ZIRKLE: Chromosome breaks by high-energy radiation are highly vari- 

 able among different kinds of cells. In newt cells we tried like the dickens to 

 break chromosomes, but got scarcely any breaks. This failure may have been 

 because with our methods we could see only relatively gross changes, but at 

 any rate, it is very suggestive. Or, maybe our calcium relations in the cult- 

 ures did not favor breaks. 



MAGEE: Does this thing include carboxylate? 



MAZIA: I don't know. Dr. Chargaff points out that we have phosphate 

 groups . 



CHARGAFF: I don't know. Of course, you can disperse nucleoproteins 

 without a chelating agent. The first modern preparations of DNA were made by 

 extracting thymus with distilled water. This way you got a very nice solution 

 of material. If the solution is brought to 0. 15 molar -- physiological saline -- 

 and precipitated, the particles can be redissolved more easily in medium 

 strength sodium chloride, '"^ut you are not sure under these conditions that what- 

 ever divalent metal was there will be left. 



MAZIA: Curt Stern revived the method of isolating nucleoprotein at low 

 ionic strength a few years ago, and we were following up his work. In the case 

 of sea urchin sperm, we could not put the nucleoproteins into solution by wash- 

 ing the nuclei in distilled water. They swelled tremendously, but nothing 

 came out. We noted that he had used arsenate to inhibit desoxyribonuclease. 

 We tried citrate, and found that after washing with citrate, we could dissolve 

 the nucleoprotein in distilled water. In our experiment, the citrate was not 

 functioning as a desoxyribonuclease-inhibitor at all, but as an agent complexing 

 divalent ions. The chelation step was necessary in the case of the sea urchin 

 and was definitely necessary when we tried to dissolve the formed, visible 

 chromosomes in the salivary glands of Drosophila and in the cells of the grass- 

 hopper testis. 



We have not worked with calf thymus. It could well be that there are 

 cases where the divalent ion bridges are less important, and that the repulsions 

 introduced by lowering the ionic strength would be adequate not only to swell the 

 chromosomes but to take them apart. 



CHARGAFF: In salt solution, you can certainly do without the chelating 

 agent, for instance, in the PoUister-Mirsky procedure. 



