Role of Preformed Structure in Cell Heredity 175 



singlet killers were crossed to kappa-free doublet sensitives; and 

 kappa-bearing doublet killers were crossed to kappa-free singlet sensi- 

 tives. 



Again, as in the experiments reported above, singlet mates produced 

 singlet clones and doublet mates produced doublet clones, regardless 

 of whether cytoplasmic transfer had occurred (Figs. 4I/i and 411/?) 

 or not (Figs. 41a and 4IIa I . Even when the bridge was broad enough 

 to see free cytoplasmic flow through it and to assure thorough mixing 

 of the cytoplasms, eventual separation of the mates, followed by their 

 reproduction, yielded clones showing the same result. Never before 

 in our experience had we encountered results suggesting the para- 

 doxical conclusion that a hereditary difference was neither genotypic 

 nor cytoplasmic in basis. Accordingly, new possibilities had to be 

 considered and subjected to analytical test. 



First of all, possibilities of nuclear determination by mechanisms 

 other than genotypic differences were considered. One such possibility 

 was nuclear differentiation. Earlier studies ( Sonneborn, 1947, pp. 293, 

 306-307) on the inheritance of mating types had shown that macro- 

 nuclei of identical genotype, arising from products of a syncaryon at 

 conjugation or autogamy, could become permanently fixed to control 

 one or the other of two alternative traits, but that this differentiation 

 persists through conjugation and autogamy when new macronuclei 

 arise by "macronuclear regeneration," i.e., by regeneration from a frag- 

 ment of the prezygotic macronucleus. This knowledge was used to test 

 whether the singlet-doublet alternative was determined by a com- 

 parable macronuclear differentiation. 



Figure 5 diagrams the design of the experiment. Matings of doublets 

 with singlets were induced to form broad cytoplasmic bridges by 

 exposure to antiserum, as set forth above. This permitted the singlet 

 and doublet mates to exchange some fragments of their prezygotic 

 macronuclei, for these are carried across by the cytoplasm when the 

 bridges are broad. In order to get the fragments to regenerate into 

 macronuclei, all of the conjugants were from stocks that were homo- 

 zygous for gene am. Homozygotes for this gene yield after conjugation 

 a high percentage of first-fission products which lack macronuclei 

 developed from the syncaryon, but which possess fragments of the 

 prezygotic macronuclei (Sonneborn, 1954b; Nobili, 1961a). The frag- 

 ments in such cells regenerate into macronuclei while segregating at 

 random to the daughter cells formed during the next several fissions. 

 These products of the fissions were isolated and grown into clones. 

 The clonal phenotypes revealed whether the macronuclei were de- 

 scended from a syncaryon (heterozygous for marker genes) or from 



