Figure 4.

PDD2 knockout cells are arrested during late stages of conjugation. (A) Western blot analysis of total proteins from conjugating wild-type (WT) and mutant (Δ) cells at different times postmixing (indicated at top in hr) resolved on a 10% SDS–polyacrylamide gel and transferred to membrane. Distinct sections of the blots were then probed with previously obtained Pdd2p antibodies (Smothers et al. 1997b) and as a loading control tubulin (α-Tub.) antibodies. Note the differences in the relative ratio between the phosphorylated (slower-migrating) and unmodified isoforms of Pdd2p between wild-type and mutant cells starting at ∼12 hr. (B) Schematic representation of the late stages of conjugation. The following stages are shown: Macronuclear Development II MAC II; Macronuclear Development III MAC III; and New Macronuclei NM. (MI)Micronucleus; (AN) anlagen; (OM) old macronucleus (black). (C) Confocal sections of wild-type (WT) and mutant (Δ) cells fixed at the indicated time points and stained with DAPI and Pdd1p or Pdd2p antibodies as shown. Asterisks (*) indicate old macronuclei, stained with DAPI. Note the size difference between old macronuclei at 11 hr, suggestive of different extents of degradation in mutant and wild-type cells. White arrowheads indicate doughnut-like DNA degradation structures that are well organized in wild-type cells but not in mutants at 13 hr. Also, note the clear presence of Pdd1p and Pdd2p in mutants at 15 hr; these proteins are not detected in wild-type cells that have eliminated one micronucleus.