68 NITROGEN METABOLISM 



percentage recombination of characters obtained in the pro- 

 geny of each cross will indicate whether or not the mutants 

 are the outcome of mutation at the same gene locus. The 

 cells of fungi are multinucleate and it frequently happens 

 that hyphae from two parents will fuse and thus form a 

 structure known as a heterocaryon which contains nuclei 

 derived from each of the parent hyphae. Consequently a 

 heterocaryon formed between two genetically identical 

 mutants will have the same growth requirements as the 

 two parent mutants. But the heterocaryon from genetically 

 different mutants will grow in the complete absence of such 

 compounds since the metabolic deficiencies of the nuclei 

 derived from one parent will be complemented by the 

 activities of the nuclei from the other parent. 



Primarily because of the ease with which they can be 

 isolated, mutants of penicillin-sensitive bacteria have been 

 used in many recent investigations [9]. Mutation is brought 

 about by irradiation with ultraviolet light in doses sufficient 

 to kill 99*9% of the organisms and then the suspension is 

 cultured in a rich medium, washed and transferred to mini- 

 mal medium containing penicillin. The non-mutants grow 

 and are consequently killed by the penicillin. The suspen- 

 sion is then plated on to a rich medium and the colonies 

 appearing are usually those of nutritionally exacting mutants. 



Having isolated a number of mutants exacting towards a 

 particular substance, other substances likely to be inter- 

 mediates in its synthesis are then tested for their ability to 

 promote grov^h, since theoretically the product of the 

 blocked reaction or any compound derived from it should 

 be active in this respect, provided the organism is permeable 

 to such substances. The sequence of intermediates in a 

 biosynthesis may then be deduced by arranging the mutants 

 in order according to the range of compounds supporting 

 grov^h. It is to be expected that the further the blocked 

 reaction from the end-product, the greater the number of 

 compounds utilized by the mutant. For example, in the 

 synthesis of D from A by: 



