278 



the mutations. For two rll mutants 

 separated by I per cent linkage dis- 

 tance (measured by a standard cross 

 on B) the proportion of mixedly in- 

 fected K yielding any wild particles is 

 about 0.2 per cent. 



This value has bearing upon the ef- 

 fect upon K/B values of the heterozy- 

 gous phage particles which arise in a 

 cross between two rll mutants on B. 

 In such a cross between closely linked 

 rll mutants, the progeny should in- 

 clude about 2 per cent of particles con- 

 taining a tra7is configuration heterozy- 

 gous piece.i^ When one of these is 

 plated on K, there is a certain chance 

 that a wild recombinant may form in 

 the first cycle of infection, leading to 

 production of a plaque. If it is assumed 

 that these are no more likely to do so 

 than a mixed infection of K with two 

 complete mutant particles, it can be 

 concluded that the efi^ect of these 

 heterozygous particles upon the count 

 on K is negligible, provided that both 

 rll mutants belong to the same seg- 

 ment. For mutants in diff^erent seg- 

 ments, however, the "efficiency" of 

 the heteozygous particles should be 

 much greater, and recombination 

 values measured by the K/B method 

 should run considerably higher than 

 the true values. The recombination 

 values in Figure 2 for crosses which 

 transgress the segmental divide are 

 probably subject to some correction 

 for this reason. 



Rough Mapping bv Spot Test.—\{ 

 a stock of either of two rll mutants is 

 plated on K, no plaques arise; but if 

 both are plated together, some bac- 

 teria become infected by both mu- 

 tants and, if this leads to the occur- 

 rence of wild-type recombinants, 



i"' Hershey, A. D., and Chase, M., Cold 

 Spring Harbor Symposia Quant. Biol. 16: 

 471-479, 1951; Levinthal, C, Genetics 39:169- 

 184, 1954. 



BENZER 



plaques are produced. If the two mu- 

 tants are such that wild recombinants 

 cannot arise between them (e.g., if 

 they contain identical mutations), no 

 plaques appear. A given rll mutant 

 may thus be tested against several 

 others on a single plate by first seeding 

 the plate with K plus the mutant in 

 question (in the usual soft agar top 

 layer) and then spotting with drops 

 containing the other rll mutants. 



Inspection of such a plate immedi- 

 ately places the unknown mutant in 

 the proper segment, since spotting any 

 mutant of segment A against any mu- 

 tant of segment B gives a very clear 

 spot, due to the extensive Ivsis of 

 mixedly infected bacteria. However, 

 for a pair of mutations belonging to 

 the same segment, plaques are pro- 

 duced only by the relatively few 

 mixedly infected bacteria which give 

 rise to wild recombinants. The greater 

 the linkage distance between the mu- 

 tations, the larger the number of 

 plaques that appear in the spot. A 

 group of mutants of the same segment 

 may thus be seriated by seeding one 

 plate with each and spotting with all 

 the others. Given a previously seriated 

 group, a new mutant can thus be 

 quickly located within the group. This 

 method works best for mutants which 

 are stable (i.e., low reversion rate) 

 and nonleaky, so that large numbers 

 of phage particles can be plated. Re- 

 versions or pronounced leaking effects 

 obviously cause an obscuring back- 

 ground. 



This test has been applied to a large 

 group of stable, nonleaky rll mutants. 

 Their approximate locations as de- 

 duced from these tests are shown in 

 Figure 4. Some of the mutants showed 

 anomalies which made it impossible to 

 locate them as members of a series. 

 They gave very litte recombination 

 with any of the mutants located within 

 a certain span, while behaving nor- 



