M. WESTERGAARD AND H. HIRSCH 



protoperithecia on the same substrates. Hence, genes and environment together 

 determine a series of different states, showing different morphological and bio- 

 chemical characteristics. 



Turning now to the second, or genetical part of the problem, it is evident that the 

 control of differentiation on a synthetic medium has been worked out sufficiently to 

 allow a study of mutants in which protoperithecial development is either blocked or 

 altered on the P-minimal medium. It has not been necessary to produce such 

 mutants experimentally. It is a well-known fact that most Neurospora strains 

 become more or less sterile when they are propagated vegetatively over a long 

 period. From such old strains we have isolated a number of mutants in single- 

 spore cultures which show various degrees of sexual sterility. Some of these mutants 

 never form protoperithecia on P-minimal medium, and they are completely female- 

 sterile (Plate lb). Fortunately, however, most of them are male-fertile so that they 

 can be studied genetically. Other mutants have abnormal-looking protoperithecia 

 which cannot be fertilized, and we have still other mutants which do develop proto- 

 perithecia and after fertilization also normal-looking perithecia, which, however, 

 never contain ascospores. The strains which do not produce protoperithecia do not 

 turn dark, confirming the evidence from the first part of the investigation, that there 

 is a relationship between the formation of protoperithecia and of melanins. We have 

 tested three of these mutants for tyrosinase activity and have found none. 



On the other hand we have a very interesting group of mutants which produce a 

 very great number of small protoperithecia ; these strains turn completely black in 

 a very short time and they are completely female-sterile (Plate Ic). Apparently both 

 absence of melanin formation and excessive melanin formation interfere with sexual 

 differentiation. Although the formal genetics of the various sterile mutants has not 

 yet been worked out, there is some evidence both from heterokaryons and from other 

 experiments that we are dealing with different, non-allelic mutants. 



If the standard biochemical genetical technique were to be applied to these mu- 

 tants it would mean feeding the different mutants with different intermediates 

 (precursors) in melanin metabolism and studying the reaction of the mycelia on the 

 supplemented media. (It is a noteworthy fact that none of the sterile mutants become 

 fertile on the standard 'complete' media, which are supplemented with vitamins, 

 casein-hydrolysate, yeast and malt extract). Unfortunately a number of difficulties 

 arise here. For one thing it is unlikely that anything would come out of feeding the 

 melanins directly to the mutants since it is unlikely that such high-molecular com- 

 pounds would penetrate the mycelium. For the same reason it seems unlikely that 

 adding tyrosinase itself to the substrate would have any effect. Unfortunately the 

 various low-molecular intermediates in melanin synthesis have not been available 

 to us (see Lerner, 1953). We have therefore tried a short-cut. Tyrosine + tyrosinase 

 prepared from cultivated mushrooms (Psalliota) were added to sterile mutants which 

 were grown for 3 days on P-minimal medium, in the hope that some of the inter- 

 mediates formed during melanin synthesis might be picked up by the mycelium. 

 Somewhat to our surprise one of the mutants (913/83) gave what might be called a 

 promising reaction to this crude treatment, forming, where substrate + enzyme was 

 added, protoperithecium-like structures (Plate Id). The induction of these structures, 

 although they were far from being normal protoperithecia, certainly suggests that 



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