Schematic diagram showing the processes occurring during macronuclear differentiation in Stylonychia lemnae. The timescale starts when the two conjugants have separated (modified from reference 1 with permission of the publisher; see also reference 25).

Southern and slot blot analyses using snRNA as a probe. (A) Ethidium bromide-stained agarose gel with total DNA (lane 1) and XbaI-digested micronuclear DNA (lane2). (B) Southern hybridization to total DNA (macronuclear and micronuclear DNA) from vegetative cells (lane 1) and to XbaI-digested micronuclear DNA (lane 2) using snRNAs from exconjugant cells at 10 h postconjugation as a probe. snRNA was isolated from the gel and end labeled as described previously (20). Hybridization was performed as described in Materials and Methods. (C) Slot blot of radiolabeled snRNAs of 10-h-postconjugation cells hybridized on different DNA sequences from Stylonychia: a, histone H4; b, calmodulin; c, rRNA genes; d, total DNA of vegetative cells; e, DNA isolated from polytene chromosomes; f, 1.3-kbp nanochromosome isolated from vegetative cells; g, pLJ01; h, 1.1-kbp and 1.3-kbp nanochromosome contained in pCE7; i, XbaI fragment of the repetitive element; j, MaA81. Each slot contains 1 μg DNA.

Effect of trichostatin A on DNA processing. (A) Inhibition of histone H3 methylation by trichostatin A; histone H3 from macronuclear anlagen from control cells or trichostatin A-treated cells. Lanes 1 and 2, Coomassie-stained gel (lane 1, control cells; lane 2, trichostatin A-treated cells); lanes 3 and 4, Western analyses using antibodies directed against trimethylated histone H3 (lane 3, control cells; lane 4, trichostatin A-treated cells). (B) Effect of trichostatin A on excision of the transposon-like element MaA81. Lane 1, molecular size marker (0.2 kbp to 10 kbp, Smart ladder; Eurogentec); lane 2, PCR product from the repetitive element of cells treated with trichostatin A at 40 h postconjugation; lane 3, control cells at 40 h postconjugation. At the top, the positions of the primers used and the amplified region (gray) are given in the schematic drawing of the repetitive element (black box). (C) Effect of trichostatin A on excision of the IESs from the 1.3-kbp MDS. Lane 1, molecular size marker; lane 2, control cells at 40 h postconjugation; lane 3, PCR product of cells treated with trichostatin A at 40 h postconjugation. At the top, the positions of the primers used and the amplified region (gray) are given in the schematic drawing of the pCE7 gene cluster (black box; IESs are indicated by white boxes within). (D) Control PCR using primers derived from the 5′ region of the 1.3-kbp nanochromosome showing that equal amounts of template DNA were present in the PCRs. Lane 1, molecular size marker; lane 2, control cells at 40 h postconjugation; lane 3, PCR product of cells treated with trichostatin A at 40 h postconjugation. At the top, the positions of the primers used and the amplified region (gray) are given in the schematic drawing of the pCE7 gene cluster (black box; IESs are indicated by white boxes within).

Isolation of RNA from vegetative and exconjugant cells at different stages of macronuclear differentiation. The RNA was separated on a denaturing polyacrylamide gel as described in Materials and Methods. The size of the snRNA synthesized during macronuclear development was estimated by using 40- to 50-nt oligonucleotides as markers. Lane 1, oligonucleotides (40 nt and 50 nt); lane 2, total RNA from vegetative cells; lanes 3 to 5, RNA isolated from exconjugants at 10 h (lane 3), 25 h (lane 4), and 50 h (lane 5) postconjugation. The RNA was isolated from approximately 10⁸ cells.

Analysis of histone H3K9 methylation in the different nuclei. (A) Western analyses using antibodies directed against histone H3 trimethylated at K9. Macronuclei and micronuclei were isolated from vegetative cells and macronuclear anlagen were isolated from exconjugant cells at 20 h postconjugation. They were separated on a 15% SDS-polyacrylamide gel, and either the gel was stained with Coomassie blue or the proteins were blotted onto a nylon membrane. Western analyses were performed as described in Materials and Methods. Lanes 1 to 3, Coomassie-stained proteins; lane 1, micronuclear proteins; lane 2, macronuclear proteins; lane 3, proteins of the macronuclear anlagen (20 h postconjugation); lanes 4 to 6, Western analysis using anti-histone H3 trimethyl K9 antibodies; lane 4, micronuclear proteins; lane 5, macronuclear proteins; lane 6, proteins of the macronuclear anlagen. Arrows point to the micronuclear histone H3 variant “X” and to histone H3. (B) Immunolocalization of histone H3 trimethyl K9 in Stylonychia nuclei at successive stages of macronuclear anlagen development. Fluorochrome channels were scanned sequentially, generating 8-bit grayscale images. False colors were assigned to each channel (histone H3 trimethyl K9, green; To-Pro-3, red) before being overlaid. (a) Maximum-intensity projections of 24 light optical serial sections showing two micronuclei (m), a macronucleus (M), and a macronuclear anlage containing polytene chromosomes (A). Histone H3 trimethyl K9 is found in micronuclei as well as in distinct areas of the macronuclear anlage but is absent in the macronucleus. (b) Mid-light optical sections of a macronuclear anlage containing polytene chromosomes (A). Histone H3 trimethyl K9 is localized in distinct areas of the macronuclear anlage (green and green-red areas). Discrete amorphous areas, possibly representing DNA already processed, contain no histone H3 trimethyl K9 (red areas). The graticule frames a segment of a polytene chromosome. In this segment, histone H3 trimethyl K9 appears to be localized preferentially in the chromosomal bands. (c) A late macronuclear anlage in the DNA-poor stage (late A). No histone H3 trimethyl K9 is found in late macronuclear anlagen in the DNA-poor stage. (C) Slot blot analysis of digoxigenin-labeled immunoprecipitated DNA isolated from exconjugants at 20 h postconjugation hybridized to different DNA sequence classes. Slots: a, macronuclear 1.1-kbp nanochromosome; b, macronuclear 1.3-kbp nanochromosome; c, macronuclear DNA; d, XbaI fragment of the transposon-like element MaA81; e, complete transposon-like element MaA81; f, repetitive sequence pLJ01; g, macronuclear nanochromosome encoding histone H4. Each slot contains 1 μg DNA.

Silencing of PIWI expression by RNAi in exconjugant cells and Western analysis using an antibody directed against histone H3 trimethyl K9. Isolation of nuclear proteins and Western analyses were performed as described in Materials and Methods and the legend to Fig. 4. Proteins of the macronuclear anlagen were isolated at 20 h postconjugation. Lanes 1 and 2, Coomassie-stained gel used for Western analysis; lane 1, proteins from cells in which PIWI expression was silenced by RNAi; lane 2, proteins from control cells; lanes 3 and 4, Western analysis using anti-histone H3 trimethyl K9 antibodies on PIWI-silenced cells and control cells; lane 3, proteins from exconjugant cells in which PIWI expression was silenced by RNAi; lane 4, proteins from control cells. Arrows point to the micronuclear histone H3 variant “X” and to histone H3.