180 GENETICS: J. LEDERBERG Proc. N. A. S. 



EMS — lactose, which, as beforeweresensitivetoTl. Taken back to EMB — 

 lactose, they showed the Lacy result of splitting off pure — and -f- types. 

 When single colonies were streaked out in series for 15 transfers on EMS — 

 lactose, they retained their normal Lac+ appearance, but throughout the 

 experiment they continued to segregate when brought on EMB — lactose, 

 which is a nutritionally "complete" medium. To summarize, H-l was 

 an unstable prototroph culture which was apparently Lac+ and Tl- 

 sensitive so long as it was maintained on a synthetic medium, but which 

 segregated nutritionally deficient, Lac— and Tl-resistants on a "complete" 

 medium. 



At this point, three alternatives had to be distinguished. The segregat- 

 ing entities might be (a) associations of intact cells of the original parental 

 mutants collaborating by syntrophism to allow growth on synthetic 

 medium; or (b) heterokaryons comprising intact nuclei of the parents in 

 the same cell; or finally (c) diploid heterozygotes. The phage sensitivity 

 of H-l , contrasted to the resistance of the parents, render (a) unlikely, but 

 the segregation might still be from a heterokaryon or a heterozygote. 

 To test these alternatives, it was thought that a heterokaryon should split 

 out each of the two parental genotypes, but no additional combinations. 

 On the other hand, segregation from a heterozygote should sometimes be 

 accompanied by crossing over, and the formation of new combinations of 

 the parental characters. 



One hundred thirty-five Lacy colonies were separately streaked out on 

 EMB — lactose, and a single pureLac+ and Lac— segregant isolated from 

 each of the 135 streakings, to insure that each of the cultures tested was 

 an independent segregant. Among the 135 Lac— tested, there were 121 

 T5 r (parentals) and 14 T5 S (exchanges); the Lac+ included 133 T5 S 

 (parentals) and 2 T5 r (exchanges). Therefore, out of 270 tests there were 

 16, or 6%, new combinations. Therefore, crossing over occurs during the 

 segregation of H-l. This is compatible only with the hypothesis of 

 segregation from a heterozygote. 



The following remarks should not obscure this major conclusion. The 

 amount of exchange in the Lac— and Lac-f- series is not the same (x 2 i = 9). 

 The crossovers in the two series came from different Lacy colonies, but 

 reciprocality cannot be demanded if many different segregations occur 

 during the growth of a single Lacy colony. Even the higher value of 

 14 /i35 or 10% f° r exchanges between Lac and Vi in the Lac— series is lower 

 than the 38% derived in previous studies on prototroph recombinants. 

 Finally, it was very obvious that the Lac— segregants far outnumbered 

 the Lac -f- . If anything, selective pressures on EMB — lactose seem to favor 

 Lac+, so this finding was most unexpected. 



Owing to technical difficulties, tests on nutritional segregations have 

 been less extensive. It proved to be especially difficult to score for the 



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