MECHANISMS OF GENE ACTION 301 



genes which have been studied act principally by their influence upon 

 enzymes. Even in mutants in which the enzyme governing the blocked 

 reaction is present, the block mav result indirectly from the inactivitv of 

 a different enzyme. 



A more general problem concerns the auxotrophic mutants themselves. 

 What fraction of all possible mutations involve blocks in particular steps 

 in biosynthesis? Attempts to answer this question experimentally have 

 involved what are called "irreparable mutants, " those which cannot be 

 kept alive by any known growth factor supplemented in the medium. 

 The frequency of this class in N eurospora has been estimated by two 

 different methods. 



One method, devised by Horowitz, employed temperature-sensitive 

 mutants, a class in which the mutant phenotype is fully expressed only 

 within a particular temperature range. The frequency of "irreparable 

 mutants was estimated by obtaining a number of mutants which would 

 grow on minimal medium at 25° C but not at 35° C. Some of these, the 

 "reparable' ones, would grow at 35° C on a supplemented medium, 

 whereas others, which would not grow on any known combination of 

 growth factors, were classed as "irreparable." In N eurospora, the 

 irreparable class amounted to 46 per cent of the temperature-sensitive 

 mutants recovered. (In £. coli, 23 per cent of the temperature mutants 

 were irreparable, but the selection method is open to criticism and will 

 not be considered further here. ) Although temperature mutants are not 

 rare among the auxotrophs, their use in this experiment is based upon the 

 assumption, stated by Horowitz, that "genes controlling indispensable 

 functions are just as likely to yield temperature-sensitixe alleles as those 

 controlling dispensable functions." This assumption is essentially un- 

 testable, but if valid, it would appear that at least half of the Neurospora 

 auxotrophs recovered after L'\'-irradiation result from some unknown 

 metabolic effect of the mutation. 



The other method of estimating the frequency of irreparable muta- 

 tions, devised by Atwood, utilized Neurospora heterokaryons composed 

 of two mutant strains, and thus consisting of two types of nuclei within 

 a common cytoplasm. Irreparable mutants are defined as those which can 

 be kept growing within the heterokarvon by the functioning of the other 

 nuclear component, but cannot grow when isolated in a monokaryon. 

 The method developed to detect these mutants is extremelv ingenious, 

 can be applied to both induced and spontaneous mutations, and makes 

 possible a very different basis for estimating indispensability than does 

 the temperature-sensitivity method. Of the U\'-induced mutants re- 

 covered by this method, over 96 per cent were found to be irreparable, 

 and of the spontaneous mutants, about 92 per cent. All of these mutants. 



