Gene Action and Polypeptides 



409 



tures to a B,-frce minimal medium would 

 produce exactly four that can grow and 

 exactly four that cannot. As expected from 

 our hypothesis, mutants are found which 

 lack B] and do not contain this final enzy- 

 matic activity. 



If for a given mutant, a number of spore 

 sacs are tested as described, the locus of the 

 mutant relative to the centromere of the 

 chromosome in which it is located can be 

 mapped (see Figure 9-10, p. 125). When 

 no chiasma occurs between the loci of the 

 mutant and the centromere, segregation of 

 normal ( + ) and mutant (th) alleles occurs 

 at the first meiotic division and — because 

 the last two divisions in the ascus are tan- 

 dem to the first — the eight ascospores occur 

 in the relative order, 



+ + + +//? th th th. 



However, when a single chiasma occurs be- 

 tween the mutant and the centromere, segre- 

 gation occurs in the second meiotic division, 

 and the ascospores occur in the relative 

 order, 



-f + th th + -f- th th. 



If a record is kept of the order of the spores 

 in each ascus, the first and second division 

 segregation arrangements can be identified 

 after the spores are grown and their geno- 

 types determined. It should be recalled that 

 if 20% of all sacs show second-division 

 segregation (two + spores alternating with 

 two th spores), then 20% of the tetrads had 

 a chiasma between the mutant and the cen- 

 tromere, and the mutant is located ten map 

 units from the centromere. 



When a number of separately-occurring 

 point mutants, defective in the enzyme which 

 catalyzes the last step in Bi synthesis, are 

 localized this way, all are found to be on the 

 same chromosome and approximately the 

 same distance from the centromere. This 

 result suggests that the catalytic ability of a 

 particular enzyme is the result of the action 

 of a particular gene. 



For the efficient detection of biochemical 

 mutants in Neurospora, certain modifica- 

 tions are made in the procedure already 

 outlined. Potentially-mutant spores are 

 grown on a medium supplemented with all 

 substances which might conceivably be in- 

 volved in biochemical mutation. Growing 

 cultures are then transferred to a basic 

 (minimal) medium containing no additions, 

 where failure to grow indicates that the mu- 

 tant culture has lost the ability to synthesize 

 some component added to the basic medium. 

 The specific ability lost is determined by 

 testing for growth in a basic medium supple- 

 mented, in turn, with the individual enrich- 

 ing components of the complete medium. 

 Techniques have been developed also to 

 eliminate nonmutant strains selectively. 

 Thus, spores given an opportunity to grow 

 for a short time on a minimal medium can 

 be subjected either to filtration, which sepa- 

 rates the larger, (growing) nonmutant cul- 

 tures from the smaller, (nongrowing) mu- 

 tant ones, or to an antibiotic which kills 

 actively-growing cultures but has less or no 

 effect on nongrowing ones. In this way, the 

 sample later tested for mutants can be mu- 

 tant-enriched. It is even possible to find 

 mutants for unknown growth factors by sup- 

 plementing the culture medium with extracts 

 of normal strains of Neurospora containing 

 various substances, both known and un- 

 known, needed by the mold. The same mu- 

 tants requiring unknown growth factors can 

 then be used in the specific assays needed 

 for the isolation and identification of such 

 substances. 



Such improvements in the techniques for 

 detecting biochemical mutants in Neurospora 

 expedite additional tests of the postulated 

 enzyme-gene relationship. Two more tests 

 are described briefly. The fiFst deals with 

 the final step in the synthesis of the amino 

 acid tryptophan and involves the catalyzed 

 union of indole (in the substrate indole- 

 glycerol phosphate) and the 3-carbon amino 



