232 Discussion 



Respiratory adaptation does not require proliferation. On the other 

 hand, all the substances showing a specific mutagenesis do produce a 

 comparable inhibitory effect on adaptation. The action of differently 

 substituted acridines is quite parallel, and the concentrations of euflavine 

 necessary to produce a half-maximal effect are very similar for mutation 

 and for adaptation (6 to 7 X 10-'m). However, the converse is not true. 

 Several substances like benzimidazole or dinitrophenol inhibit adaptation 

 without producing mutants. It should be added that Harris in our 

 laboratory found that a continuous anaerobic culture for about a hundred 

 cellular generations neither increases the percentage of "petites" in the 

 population nor diminishes the capacity to form cytochrome oxidase 

 adaptively (Harris, M. (1956), J. cell. comp. Physiol., 48, 95). 



Fulton: How did you measure the cytochrome oxidase present? 



Slonimski: The method we use routinely is as follows: we make an 

 extract of cells, spin down the so-called granules, then either we measure 

 the oxygen uptake in the presence of the hydrogen donor, which is 

 ascorbic acid, and in the presence of a saturating amount of cytochrome 

 c (more precisely in the presence of 4 different concentrations of cyto- 

 chrome c and extrapolate to saturation); or we measure spectrophoto- 

 metrically the rate of oxidation of reduced cytochrome c. This is quite 

 laborious. Not all the experiments were performed in this way, the 

 majority of the experiments were performed by measuring the rate of 

 overall respiration of intact cells under the conditions where we have 

 shown previously that it is proportional to the amount of cytochrome 

 oxidase, as measured by the first method. 



Fulton: Your first method requires a lot of material, and your second 

 one probably less ? 



Slonimski: Much less. 



Fulton: Had you any trouble in reducing cytochrome c? 



Slonimski: We had a little difficulty at the beginning when we used 

 palladium. 



Fulton: We have always experienced trouble in recovering pure reduced 

 cytochrome c, after reduction with palladium, unless the metal is removed 

 in an inert atmosphere, and the method is time-consuming. 



Slonimski: The most satisfactory method for reduction is, in my 

 opinion, the one introduced by Chantrenne (1955, Biochim. biophys. 

 acta., 18, 58). It consists in reducing cytochrome c by passing it on 

 Duolite S-10, treated previously with Na2S204. We used this method 

 with commercial cytochrome c which contains some impurities. 



Fulton: It tends to undergo auto-oxidation. 



Slonimski: If you have peroxides, yes. However, the cytochrome c 

 reduced on the ion exchange resin can be kept reduced for months if 

 frozen. I personally prefer the first manometric method, although it 

 requires a lot of material; but with yeast we had no trouble. 



Davis: Have you tried the mutagenic action of your compound on 

 more ordinary mutations? 



Slonimski: We have already started some experiments on two things: 

 one is mitotic crossing over in yeast and the second is what is called 

 gene conversion (non-reciprocal recombination). 



