MUTAGENESIS 129 



what is known from microorganisms and would certainly be very 

 interesting. 



To return then to mutagen specificity in microorganisms. I am not 

 going to deal with forward mutations — I did that yesterday. Heslot's 

 data are the only relevant ones I could find on this point. 



At the same conference in Gatersleben where Heslot (35) presented 

 these data, Westergaard (78) gave data on forward mutations in 

 Penicillium, and these have been published in the same Symposium 

 volume. Westergaard did not find any specificity and he did, in fact, 

 not expect any. He classified the mutants phenotypically, by their 

 requirements, and one would hardly expect this to result in specificity. 



I shall confine my discussion to reverse mutations from auxotrophy 

 to prototrophy, and I want to describe the technique of these experi- 

 ments because this happens to be very important. Let us take an 

 example. Let us assume we screen for tryptophan reversions in either 

 Neurospora or bacteria. First, one grows the organisms in or on a 

 certain medium, then one makes a suspension, which is then distributed 

 into two tubes. One tube is treated, the other is the control. After 

 treatment one plates, which is a procedure taken too much for granted. 

 Let us look at it a bit more closely. There are two sets of plates, one for 

 determining viability, the other for determining mutation frequency. 

 Both of these are important for the calculation of mutation frequencies, 

 for in bacteria always, and in Neurospora usually, mutation fre- 

 quencies are calculated per survivors. Even if they are calculated per 

 numbers plated, this number is derived from the colonies formed on 

 the viability plates, not from direct counts of cells treated. It is im- 

 portant to keep in mind that the two sets of plates differ, and they 

 differ in two respects. In the particular example chosen, the viability 

 plates have tryptophan, the mutation plates have not. The other dif- 

 ference is that the viability plates are plated at low density, say one 

 hundred to a few hundred cells per plate, while the mutation plates 

 are plated at very high densities, say 10^ or 10^ per plate. This is 

 something I want you to keep in mind. 



Now to come to causes, possible causes of specificity. The first one 

 occurs at the stage where the mutagen has to penetrate, has to make 

 its way through the cytoplasm and through the gene-associated protein 

 in order to produce a reaction with the DNA. This may already pro- 

 duce what appears as specificity. Of course, one tries to compare 

 different mutations on an isogenic background; that is to say, one 

 preferably would compare mutations which have arisen spontaneously 

 in the same strain. But one should not overlook the possibility that 



