Vol. 35. 1949 GENETICS: J. LEDERBERG 181 



biotin requirement in the presence of amino acids, probably due to partial 

 replacement. The classification of the thiamin requirement (Bi — ) also 

 requires fastidious attention to clean chemicals and glassware. Most of 

 the Lac-f-T5» segregants are the parental B — M— ; Lac — T5 V are usually 

 T — L — B i — . However, prototrophs and the multiple mutant combinations 

 M — T — L — and M— T — L — Bi— have been encountered . These combina- 

 tions could not be obtained by previous methods, which necessarily iso- 

 lated prototrophs. 



Unstable heterozygotes like H-l had not been encountered before, 

 although many hundreds of Lac+ prototrophs from crosses heterozygous 

 for Lac had been streaked out on EM B — lactose. The persistence of H-l 

 as a diploid might be a non-genetic accident or perhaps an effect of a spon- 

 taneous life cycle mutation. If so, it would be possible to set up crosses 

 to yield diploids heterozygous for additional markers. 



Three segregants from H-l have been used in crosses to test for the 

 production of persistent heterozygotes. (A) (W-466) was B — M — Laci — 

 Vi 5 ; (;B) (W-477)T-L-B 1 -Laci- Vi r and(C)(W-478)B-M-Lac + Vi 5 . 

 These cultures were crossed on EMS— lactose with appropriate, "standard" 

 complementary stocks (B — M— orT— L — Bi— ; Lac — orLac-f-)ofindepen- 

 dentorigin. Lac+ prototrophs were streaked out on EMB — lactose, and Lao> 

 colonies looked for. In each of these crosses, about 5% of the Lac+ 

 prototrophs were heterozygous. Crosses of A X C gave the same result. 

 If "Het" is located on a chromosome, it is effective whether present in 

 one or both parents. 



In further studies still in progress, C has been crossed with multiple 

 fermentation mutant stocks, T — L — Bi — La^ — Mai — Gal — Ara — Xyl— , 

 and some also Mtl — . Such stocks were developed by irradiations 

 on EMB — media, in sequence. As before, about 5 to 10% of the + proto- 

 trophs isolated from EMS lactose or xylose plates are heterozygous. 

 However, these cultures are not uniformly heterozygous for all the factors 

 in which the parents differed. It is especially notable that in over a 

 hundred heterozygotes obtained in this way, Mai has never been hetero- 

 zygous. Usually, the cultures are pure Mai — , sometimes Mal + . Xyto 

 cultures are equally often Lacw or pure for Lac, as well as the converse. 



It remains to be decided whether the "pure" characters are homozygous 

 or hemizygous. The Mai locus which, as mentioned, has always been 

 "pure" is probably hemizygous, as shown by reversion studies. If the 

 cultures were homozygous, Mai— would be twice represented, i.e., 

 Mai — /Mai — . Reverse mutations of one of these genes should lead to 

 heterozygosity at the locus. Preliminary tests on reversions for Mai in 

 these stocks have invariably given pure Mal+ cultures, although they still 

 segregated for other factors. This suggests that Mai is represented only 

 once, that it is hemizygous, and, therefore, that there is deficiency for a 



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