Figure 3.

Disruption of the PDD2 gene eliminates parental expression during early stages of conjugation. (A) Schematic representation of the gene-disruption construct. The open box represents the coding region of the PDD2 gene. The hatched box is the coding region of the neo gene. The speckled box is a H4 histone promoter, and the solid box is a Btu2 terminator. A 1.2-kb PDD2-specific probe is shown at bottom. (B) Total genomic DNA (∼3μg) was digested with Hind III and probed by Southern blot hybridization with the probe shown above. The strains from which DNA was used are indicated at top. (C) Total cellular RNA isolated from wild-type (WT) and mutant (Δ) cells at the indicated time points was probed in Northern blot hybridization with the PDD2-specific PCR fragment (indicated in A, top), or as a loading control, with 28S ribosomal DNA (bottom). (D) Wild-type (WT) and mutant (Δ) cells fixed at 6 hr postmixing and stained with DAPI (top) or with antibodies against Pdd2p or Pdd1p (bottom). Note that the absence of Pdd2p-specific signal in PDD2 knockout cells cannot be attributed to a fixation artifact, as cells from the same source stain positively when probed with Pdd1p antibodies.