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CHAPTER 32 



chemical mutation. Growing cultures are 

 then transferred to basic (minimal) medium 

 containing no additions, where failure to 

 grow indicates a mutant culture which has 

 lost the ability to synthesize some component 

 added to the basic medium. The specific 

 ability lost is determined by testing for growth 

 in basic medium supplemented in turn with 

 the individual enriching components of the 

 complete medium. Techniques have been 

 developed also to selectively eliminate non- 

 mutant strains. Thus, spores given an oppor- 

 tunity to grow on minimal medium can be 

 subjected either to filtration, which separates 

 the larger (growing) nonmutant cultures from 

 the smaller (nongrowing) mutant ones, or 

 to an antibiotic which kills actively growing 

 cultures, but which has less or no effect on 

 nongrowing ones. In this way, the sample 

 later tested for mutants can be mutant- 

 enriched. It is even possible to find mutants 

 for unknown growth factors by supplement- 

 ing the culture medium with extracts of nor- 

 mal strains of Neurospora which contain 

 various substances, both known and un- 

 known, that are needed by the mold. The 

 same mutants requiring unknown growth 

 factors can then be used in the specific bio- 

 assays needed for the isolation and identifica- 

 tion of such substances. 



Such improvements in the techniques for 

 detecting biochemical mutants in Neurospora 

 expedited additional tests of the enzyme- 

 cistron relationship. Two additional ex- 

 amples will be described briefly. The first 

 study deals with the final step in the synthesis 

 of the amino acid tryptophan, which involves 

 the catalyzed union of indol and the 3-carbon 

 amino acid serine by the enzyme tryptophan 

 synthetase. First, separately occurring trypto- 

 phan-requiring point mutants were obtained; 

 of these, the only ones tested were those that 

 proved to be blocked in the final synthetic 

 step and were lacking full tryptophan syn- 

 thetase activity. Of 25 mutants qualifying, all 

 proved to be located on the same chromo- 



some at about the same locus. The second 

 example involved the final step in the syn- 

 thesis of adenine which is catalyzed by the 

 enzyme adenylosuccinase which splits succinic 

 acid off adenylosuccinic acid to leave adenine. 

 Of 137 independently occurring point muta- 

 tions with little or no adenylosuccinase 

 activity, again, all were on a particular chro- 

 mosome at about the same locus. The cis- 

 trons acting to specify tryptophan synthetase 

 and adenylosuccinase are different, each hav- 

 ing completely separate locations in the 

 genome. 



These results, and similar ones for other 

 enzymes in Neurospora, offer strong support 

 for the hypothesis that the specificity of all 

 enzymes is under cistronic control. More- 

 over, different enzymes are specified by dif- 

 ferent cistrons. The fact is observed that 

 the addition of Bi, tryptophan, or adenine 

 to the diet of mutants defective in the en- 

 zymes directly responsible for their respec- 

 tive syntheses makes the mold completely, 

 or almost completely, normal. This is good 

 evidence that the cistrons involved have only 

 one function to perform, which is to deter- 

 mine the specificity of one enzyme. If a 

 cistron had more than one primary effect, 

 surmounting one defect nutritionally would 

 not be expected to produce normality or near- 

 normality in all cases. From the results so 

 far described, we might also be tempted to 

 conclude that the total specificity of an en- 

 zyme is the result of the primary action of a 

 single cistron, because in all these cases the 

 enzymatic defect was found to be due to a 

 defect in one specific localized area of the 

 genetic map. We can call this the one enzyme- 

 one cistron hypothesis. 



All enzymes are protein, at least in part, 

 and the specificity of an enzyme is known 

 to be due to its protein content. Proteins 

 are composed of amino acids (Figure 32-2) 

 strung together in chains to form polypep- 

 tides, so that the specificity of an enzyme 

 must be attributed to the number and kinds 



