282 



CHAPTER 32 



elude those which can still make Bi them- 

 selves, and those which can no longer make it, 

 but which obtain Bi from the culture medium. 

 Once the spores have grown sufficiently, a 

 portion of each of the haploid growths can 

 be placed on the basic, minimal medium, 

 which contains no Bi. Those samples that 

 continue to grow when transplanted to mini- 

 mal medium are presumably nonmutant, in 

 respect that they can perform all the biochemi- 

 cal steps leading to Bi production; those 

 samples that fail to grow presumably carry 

 mutants involving failure to make Bi. Those 

 failing to grow might be mutants which lack 

 a precursor of Bi, a precursor whose presence 

 as such is not essential for growth, but which 

 is essential for subsequent Bi synthesis. Any 

 mutant of this type can be eliminated from 

 further consideration if it grows on minimal 

 medium supplemented with the particular 

 thiazole and pyrimidine which are the imme- 

 diate precursors of vitamin Bi. (All other 

 imaginable nutritional factors except Bi itself 

 may also be added, but they would have no 

 effect on the decision being made.) The 

 residue of cultures, which grow on medium 

 containing Bi but not on medium containing 

 the immediate precursors of Bi, are clearly 

 defective for the enzyme that catalyzes the 

 last step in Bi synthesis. 



Each of the haploid strains remaining is then 

 crossed to a haploid strain normal for Bi 

 synthesis, and each diploid hybrid is studied 

 separately as follows. After the hybrid under- 

 goes meiosis (refer to pp. 121-124), a sac 

 containing eight haploid ascospores is pro- 

 duced. Each of the eight spores is removed 

 and grown on Bi-supplemented minimal 

 medium. If the haploid strain being tested 

 is indeed Bi-deficient because of a mutant, 

 a subsequent transplant of each of the eight 

 haploid cultures to Bi-free minimal medium 

 will produce exactly four that can grow and 

 exactly four that cannot. Note that because 

 all four products of meiosis are recovered 

 and tested, each in duplicate, the 4 : 4 ratio 



is purely mechanical and is not subject to 

 errors of sampling, as it would be if a random 

 sample of spores were taken from a large 

 pooled lot made up by mixing the spores from 

 many sacs. When the experiment was per- 

 formed, mutants were found. These had 

 changed the specificity of the enzyme studied, 

 as expected on our hypothesis. 



If, for a given mutant, a number of spore 

 sacs are tested as described, it is also possible 

 to map the location of the mutant relative 

 to the centromere of the chromosome in 

 which it is located. The way in which this 

 is determined is illustrated in Figure 32-1, 

 which shows only the single pair of chromo- 

 somes involved. If, as shown in the left 

 portion of the Figure, no chiasma occurs be- 

 tween the loci of the mutant and the centro- 

 mere, segregation of normal (+) and mutant 

 {th) recons will occur at the first meiotic divi- 

 sion, and, because the last two divisions in 

 the ascus are tandem to the first, the eight 

 ascospores will be in the relative order 



+ + + + //2 ?/7 th th. 

 If, however, a single chiasma does occur 

 between the mutant and the centromere, as 

 shown in the right portion of the Figure, 

 segregation will occur in the second meiotic 

 division, and the ascospores will be in the 

 relative order 



+ + th th + + th th. 

 If a record is kept of the order of the spores 

 in each ascus, then these two segregation 

 arrangements can be identified when the spore 

 genotypes are determined later. If 20% of 

 all sacs showed second division segregation 

 (two + spores alternating with two th spores), 

 then 20% of the meiotic divisions had a chi- 

 asma between the mutant and the centro- 

 mere. This, you remember, means that the 

 mutant is located 10 map units from the 

 centromere. 



When a number of separately occurring 

 point mutants, defective in the enzyme cata- 

 lyzing the last step in Bi synthesis, were local- 



