1. Volume 4, Issue 5, Pages 865-872 (November 1999)
Parental Expression of the Chromodomain Protein Pdd1p Is Required for Completion of Programmed DNA Elimination and Nuclear Differentiation 
Robert S Coyne, Mikhail A Nikiforov, James F Smothers, C.David Allis, Meng-Chao Yao 
Molecular Cell 
Volume 4, Issue 5, Pages (November 1999)
DOI: /S (00)

2. Figure 1 Pdd1p Localizes to Nuclei Early in Development
Mating Tetrahymena from 2.5 hr (A and B) or 4.5 hr (C–E) were processed for either staining with DAPI (A and C) and immunocytochemistry using α-Pdd1p antibodies (B and D) or thin-section immunocytochemistry using α-Pdd1p antibodies (E). Closed triangles: somatic macronuclei. Open triangles: germline micronuclei. Arrows in (E): electron-dense structures at the periphery of a somatic macronucleus containing the majority of α-Pdd1p immunogold signal (bar in [D] = 5 μm; bar in [E] = 1 μm).
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2
Pdd1p Appears Later and Persists Longer in the Somatic Knockout Mating
Whole cell proteins of either a wild-type or pdd1::neo mating were separated by SDS-PAGE (6%) and immunoblotted. Pieces of the filter were probed with either anti-Pdd1p, anti-Pdd3p (M. A. N. and C. D. A., unpublished results) or anti-α-tubulin antiserum. (A) Comparison of developmental expression patterns. (B) Comparison of Pdd1p quantity: equivalent amounts of protein from 10 hr wild-type or knockout matings were electrophoresed in adjacent lanes and processed as described above. Signal intensity was quantified using NIH Image.
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3 Disorganized Localization of Pdd1p and Pdd2p
(A and B) At 12 and 14 hr, cells were stained with either anti-Pdd1p or anti-Pdd2p antiserum and counterstained with DAPI to visualize the anlagen (indicated by arrows), the adjacent, smaller micronuclei, and (to the left in the 12 hr images) parental macronuclei. (B) shows serial confocal sections through 14 hr anlagen. (C) shows electron microscopic examination of 14 hr conjugating cells. Arrows indicate electron-dense regions characteristic of Pdd1p-containing structures. Bar = 1 μm.
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4
Delay in Final Nuclear Event and Block in DNA Endoduplication in pdd1::neo Mating
(A) Mating cells were stained with DAPI to reveal their nuclear stage. MAC II: mating partners still paired, anlagen swelled. MAC III: pairs separated, two micronuclei present. NM (new MAC): one micronucleus remaining.
(B) Wild-type (WT) and mutant (pdd1::neo) cells were lysed, stained with propidium iodide, and the relative DNA contents of their nuclei measured by flow cytometry. Ploidy was determined from standards of micronuclei purified from starved cells (Allis and Dennison 1982).
Molecular Cell 1999 4, DOI: ( /S (00) )

6. Figure 5
Absence of Parental Pdd1p Disrupts DNA Elimination but Does Not Detectably Disrupt Chromosome Breakage
(A) Schematic of DNA elimination assay. Thin lines: DNA retained in the macronucleus. Open box: a deletion element. Arrows: oligonucleotide primers.
(B) Deletion assay results. MW: size markers. wt: product from a wild-type control. 1–8: products from pdd1::neo exconjugants. Expected size of unrearranged products: filled arrowheads; correctly rearranged products: open arrowheads.
(C) Schematic of chromosome breakage assay. Open box: chromosome breakage sequence (Cbs). Shaded boxes: telomeric repeats.
(D) Breakage assay results. Each pair of lanes shows products of two reactions on a single exconjugant. Each “I” lane shows control amplification of micronuclear-specific DNA (top reaction in [C]). Each “A” lane shows the presence of correctly broken and telomerized chromosome ends (bottom reaction in [C]). 819, etc., identify the breakage sites tested (Yao et al. 1987).
Molecular Cell 1999 4, DOI: ( /S (00) )