GENETIC RECOMBINATION IN ESCHERICHIA COLI. By Joshua Lederberg A Dissertation Presented to the Faculty of the Graduete school of Yale University in Candidacy for the Degree of Doctor of Philosophy 1947 ABSTRACT Wild type or "prototroph" cells were found in a proportion ca. Lo76 1n mixed cultures of biochemical mutants of E coli. It could be shown that these did not result from spontameous bacx-mutation. In addition th wild types, other recombination types were found suggesting the occurrence of a sexual process in E. coli. The analysis of the segregation of factors for lac- tose fermentation and for resistance to bacteriophage led to the conclusion that a single linkage group was present, on which eight factors Rave been mapped. Experiments confirming the linear order of genes, and bipa- rental inheritanee were performed. It was concluded that E. coli is normally haploid and that 1t undergoes sexual fusion with in- mediate reduction-division, during which crossing-over takes place. The alternative explanation that the apparent recombinations are due to soluble transforming factors is discussed in detail and shown to be uneconomical. Due to the rarity of the process cytological examination was unfeasible. Attempts to reveal recom- bination in two other strains of E. coli were unsuccessful, as were attempts to induce aberrations in the chromosomal arrangement or the ploidy of the species. ACKNOWLEDGMENTS The author is pleased to acknowledge the cordial assistance and cooperation given him by numerous indivi- duals, only some of whom can pe named. They include: Dr. M. MeCarty, for a generous sample of desoxyribonuclease. Drs. S.E.turia and M.Demerec for cultures of bacteriophages. Dr. E. Witkin for E. coli strain B/r. pr. R.R.eRoepke for E, coli I-15, and mutants thereof. Dr. H. Zelle, for single“eell isolations of recombinants. Dr. K. Mather (John Innes Hort. Inst.) for indispensable suggestions and criticism of the linkage analysis. Many other participants of the 1946 Cold Spring Harbor Symposium on the genetics of microorganisms for their valuable advice and encouragement. He is particularly indebted to Prof. F.Jd. Ryan for having introduced him to the subject of the genetics of microorganisms, © ana for having facilitated his association with Prof. 4.1L. Tatum. To. Prof. Tatum,this final mention is reserved, as an ina- dequate measure of the author's indebtedness and gratitude for guidance, active cohlaboration, encouragement, and physical support of the investigations herein reported. The author was a Fellow of the Jane Coffin Childs Memorial Fund,for Medical Research. “his work was supported by a grant from the Jane Coffin Childs Memorial Fund. CONTENTS Introduction Review of literature: sexuality : transformation : "zygotes" Material and Methods Prototroph recombinants, recovery Results and Conclusions Spontaneous mutation Prototrophs Syntrophisn Attempts to extract transfor- ming factors Nutritional recombinations Lac, Vy segregations Crossover theory Linearity; Ve segregation Attempts to induce aberrations Four-strand crossing over Attempts to induce diploidy Bi-parental inheritance and transformation Other E coli strains Discussion BibiLLlographny. WwW Mme HF F bacterial variation 7 11 13 14 15 17 19 21 26 29 32 33 35 37 40 4) De o. Index tc Tables Summary of mutants A. Spmbois Used. B. Derivation of mutant stocks. Biochemical recombination types found in a mixed culture of Y-24 and Y-46. Comparisons of v,* segregations with altermative parental couplings. The segregation of Lac and Vy- A. Data of individual experiments. Lac, Vi Scores on colonies isolated from minimal agar plates in the cross Y-40 x Y~53. B. Data of individual experiments. Lac, Vj, scores on colonies isolated from thiamin-supplemented plates in the cross Y-40 X Y-53. Cc. Data of individual experiments. Lac, V, scores on colonies isolated from minimal agar in the cross Y-64 X 58-Lol. D. As above. Scores on colonies isolated from thiamin- supplememted agar. E. Lac, V, scores on colonies isolated from minimal agar an the dross Y-87 X Y-10. F. AS above. Isolates from thiamin-supplemented agar. G. Tests of allelism of mutations at Lac and Vy loci. Summary of Taples 4. Data of Table 5 rearranged to bring corresponding inter- change classes in the same column. Relative frequency of various biochemical recombination classes in the cross B-M-T+L+B, + X B+tM+T-L-B~. - Segregation of Lac, Vy and Ve. Pairwise occurrence of recombination in mixtures of three components. Figure 1. Phenotypes of combinations of Lac and Vy on EMB-lactose agar. V,¥ mutations from V,*.