Extended Data Figure 8: Genetic and molecular analysis of the cross between strains 227 and 38 of P. septaurelia.

a, Maternal inheritance of mating types in the wild-type strain 227. b, Strain 38 is genetically restricted to O expression, but constitutively determined for E. The mutation identified in strain 38 is of particular interest because it affects both the expression and the inheritance of mating types, which suggests that it lies in a gene that controls mating-type determination through an alternative rearrangement. Indeed, known P. tetraurelia mutations fall in two distinct categories. mtA^O, mtB^O and mtC^O prevent expression of type E but have no effect on the rearrangement that determines mating types or on its maternal inheritance, whereas mtF^E lies in a trans-acting factor required for a subset of rearrangements during MAC development but has no effect on the expression of mating types during sexual reactivity. The only type of mutation that can be envisioned to affect both expression and determination/inheritance would be a mutation preventing expression of a functional protein required for E expression, and at the same time preventing in cis (by destroying a potential Pgm cleavage site) the rearrangement that normally inactivates this gene in the MAC of wild-type O cells. The mt^XIII allele of strain 38 restricts cells to O expression in a recessive manner, but also has a maternal effect that enforces constitutive E determination in sexual progeny. Notably, elegant experiments showed the latter effect to be dominant²⁰: the sexual progeny of a cell carrying at least one mt^XIII allele can never be determined for O or transmit O determination. c, Sequencing of the mtB³⁸ allele revealed features that may account for both effects. A frameshift mutation makes it a pseudogene, explaining the genetic restriction to O expression. In addition, a 6-bp deletion removes one of the IES-like boundaries used in the mtB²²⁷ allele for coding-sequence deletions in O clones. Given the requirements of IES excision in P. tetraurelia and in sibling species³⁶, this can be predicted to make the same deletions impossible in the mtB³⁸ allele, which would explain the constitutive E determination effect. The full-length mtB³⁸ pseudogene in the MAC of 38 cells would indeed be expected, after a cross to 227, to protect the highly similar zygotic mtB²²⁷ allele against coding-sequence deletions in the derived F₁, through titration of homologous scnRNAs. d, Molecular analysis of the 38 × 227 cross. To verify this maternal effect, we crossed an E-expressing 227 clone (pmtB⁵¹-transformed clone T, same as in Fig. 5b; C, uninjected control) with strain 38, and F₁ heterozygotes were tested for mating types and for mtA expression by northern blotting. As expected, F₁ heterozygotes deriving from the 38 parent (1b and 2b, as determined by sequencing of a mitochondrial polymorphism) were E and expressed mtA, indicating that the incoming mtB²²⁷ allele had been rearranged into a functional, full-length form in the MAC. After autogamy of F₁ clone 2b, 24 independent F₂ homozygotes were isolated and tested for mating types. Consistent with the Mendelian segregation of mtB alleles, 12 were O and 12 were E (Supplementary Table 5); northern blot analysis of 3 clones of each type showed that only E clones expressed mtA. e, All F₂ clones maintained the full-length mtB gene in the MAC. PCR11 (Supplementary Table 6) amplifies an 888-bp fragment from the MAC version of mtB³⁸, and an 893-bp fragment from the full-length MAC version of mtB²²⁷. C1 and C2, control PCR11 on two O clones of strain 227, showing the 806-bp and 744-bp fragments resulting from the two alternative coding-sequence deletions. f, Mating types co-segregate with mtB alleles among F₂ homozygotes. Digestion of the PCR products with AluI distinguishes the 38 and 227 alleles. mtA and mtC alleles segregated independently (Supplementary Table 5).