Characterization of ΔEMA1 cells. (A) EMA1 locus and KO construct. A part of the EMA1 coding sequence, including the conserved helicase domains (shown in gray) was replaced by the drug-resistance marker neo3. Upon transformation, the KO construct was introduced into the EMA1 locus by homologous recombination. (B) Southern hybridization of ΔEMA1 strains. Total DNA isolated from wild-type (WT) or ΔEMA1 strains was digested with NdeI (N in A), and the blot was hybridized with the probe shown in A. Positions of the bands for wild-type and KO loci are indicated with arrowheads. (C) Ema1p expression in ΔEMA1 strains. (Top panel) Ema1p expression in the wild-type (W) and ΔEMA1 (Δ) strains in starved (S) and mating (4, 8, and 12 h post-mixing) cells was analyzed by Western blot using anti-Ema1p antiserum. (Bottom panel) For a loading control, the amount of α-tubulin was analyzed. (D) Developmental profiles of conjugation in wild-type and ΔEMA1 strains. Conjugation stage wild-type (CU427 × CU428) and ΔEMA1 [(7)-3-1 X (8)-1-1] cells were observed by DAPI staining. The stages categorized were single unmated cells (S), premeiosis (E1), meiosis (E2), prezygotic (M1), post-zygotic (M2), Mac development (L1), Pair Separation (2 Mics) (L2), and Mic elimination (L3). See Supplemental Figure S1 for the developmental stages. (E) ΔEMA1 causes arrest at Pair Separation stage. At 36 h post-mixing, the progeny of wild-type (CU427 and CU428) or ΔEMA1 cells were fixed and nuclei were observed by DAPI staining. The stages categorized were the same as in D except stages E1∼L1 were combined. (F) ΔEMA1 cells fail to produce viable progeny. At 6∼8 h post-mixing, single mating pairs were placed into drops of medium and incubated for ∼60 h at 30°C. Completion of conjugation was confirmed by testing for expression of the marker specific for newly developed Macs.

Association of Twi1p with chromatin in ΔEMA1 cells. (A–E) Cells with partially spread chromatin from wild-type (A,B,D) or ΔEMA1 (C,E) strains were fixed at the indicated time points post-mixing (mating). The localization of Twi1p (middle column; green in the right column) was analyzed by immunostaining using an anti-Twi1p antibody. DNA was counterstained with DAPI (left column; purple in the right column). (S) Single, nonmating cell; (P) paired mating cells; (CS) chromatin from a single cell; (CP) chromatin from paired cells. Arrows in B–E indicate chromatin from mating cells. In this experiment, the cell cortex (blue asterisks) has background staining for anti-Twi1p.

Expression of scnRNAs in ΔEMA1 cells. Total RNA was extracted from starved (S) or conjugating (2, 4, 6, 8, 10, and 12 h post-mixing) wild-type (W) or ΔEMA1 (Δ) strains and separated in sequencing gels. (A,B) Bulk scnRNAs (∼28 nt) were visualized by staining the gels with GelRed. (C–K) Blots were hybridized with the probes indicated (right). Quantitation of the signals are shown as ratios of the signals (ΔEMA1/wild-type) obtained from gel staining and Northern blots.

A refined scnRNA model. The sequentially occurring events are drawn from top to bottom. The approximate stages when the events occur are shown on the right with arrows. See the text for details.

Identification and characterization of EMA1/Ema1p. (A) Conjugation. In the sexual process of conjugation, two cells mate (1); the Mic undergoes meiosis to form two haploid pronuclei, one of which is reciprocally exchanged between the two conjugating cells (2); the migratory and stationary pronuclei then fuse to create a zygotic nucleus (3); the zygotic nucleus divides mitotically twice to produce the next generation of new Macs and Mics (4); paired cells separate and one of the two new Mics and the parental Mac are destroyed (5); and cells resume vegetative growth with Mic mitosis, followed by cytokinesis (6). (B) Copurification of Ema1p with Flag-HA-Twi1p. Two wild-type strains (No-tag) or two Flag-HA-TWI1 strains were mated and were harvested at 9 h post-mixing. Flag-HA-Twi1p-containing complexes were enriched by gel filtration and immuno-affinity purified first with anti-Flag antibody and then with anti-HA antibody. The purified proteins were separated by SDS-PAGE and analyzed by silver staining. The arrow and the asterisk indicate the positions of Flag-HA-Twi1p and the Twi1p-associated proteins identified by mass spectrometry analysis, respectively. (C) Twi1p coimmunoprecipitates with Ema1p. An EMA1-HA strain and a wild-type strain were mated and lysed at 6 h post-mixing. (Right) The Ema1p-HA-containing complex was pulled down with an anti-HA antibody. (Left) As a control, two wild-type strains were crossed and processed similarly. Coimmunoprecipitated proteins (anti-HA IP) or total proteins (Input) used for immunoprecipitation were analyzed on Western blot using anti-Twi1p antiserum. (D) Expression of EMA1 mRNA. Total RNAs from exponentially growing (E), starved (S), and conjugating (2, 4, 6, 8, 10, 12, and 14 h post-mixing) wild-type cells were analyzed by Northern hybridization. RPL21 was used as a loading control. (E–L) Localization of Ema1p. Mating pairs of wild-type cells in the early (E) and late (F) premeiosis, pronuclear exchange (G), Mac Anlagen (H,I), Nuclear Alignment (J), Pair Separation (K), and Mic Elimination (L) stages were processed for immunostaining. See Supplemental Figure S1 for the conjugation stages. Ema1p was localized using anti-Ema1p antiserum (left) and DNA was stained by DAPI (right). Arrows, arrowheads, and arrowheads marked with “An” indicate Macs, Mics, and developing new Macs, respectively. The staining detected at the junction of cells (double arrowheads) was also observed in ΔEMA1 strains (see Fig. 7O,P) and thus represents cross-reaction of the antiserum with other proteins.

DNA elimination in the progeny of ΔEMA1 cells. (Left) Schematic drawings of the IES elimination assays. Solid horizontal lines indicate MDSs and the open boxes indicate IESs. The M and R elements are ∼2.5 kb apart on the same Mic chromosome. Four primers (arrows) that flank each IES were used for nested PCR. For the assays of Cal and Tlr-1 elements, primers complementary to IESs were also used for the same PCR. (Right) Results of the IES elimination assays. The sizes of the unprocessed (Mic form) and the processed (Mac form) products are marked by arrowheads with “i” and “a”, respectively. (m) Molecular weight marker.

Three types ncRNA. (A–D) Exponentially growing (E), starved (S), and conjugating (2, 4, 6, 8, 10, and 12 h post-mixing) wild-type cells were analyzed by RT–PCR. (A) Schematic drawing of the analysis of ncRNA transcription from the parental Mac. Sets of primers (arrows) that flank four different IESs were used to amplify the cDNAs. (B) Results of RT–PCR for Mac loci flanking the indicated elements. The red horizontal bar indicates stages in which the ncRNAs are up-regulated. Constitutively expressed RPL21 mRNA was amplified as the positive control for the RT reaction. (C) Schematic drawing of ncRNA analysis from IESs. Sets of primers (arrows) in four different IESs were used to amplify the cDNAs. (D) The results of RT–PCR for the indicated IES elements. Blue and green horizontal bars indicate stages in which the ncRNAs are up-regulated. (E) Schematic summary of ncRNA transcription analyses. Relative expression levels (vertical axis) and expression timing (horizontal axis) of the ncRNAs from the Mic (blue), the parental Mac (red), and the developing Mac (green) are represented. (F) Coimmunoprecipitation of ncRNAs with Twi1p. (Lanes labeled as A) Conjugating wild-type strains at 4.5 or 9 h post-mixing were lysed and the Twi1p complex was immunoprecipitated with anti-Twi1p antibody. Coimmunoprecipitated RNA was analyzed by RT–PCR (RT+) to detect M-MDS (from parental Mac) or M-IES (from developing new Mac) transcripts as above. As a control, a similar experiment was performed with preimmune serum (lanes labeled as P). In parallel, a similar experiment was performed without the reverse transcription reaction (RT−). (G) Coimmunoprecipitation assay of ncRNAs with Twi1p in the wild-type (W) or ΔEMA1 strains (Δ). Conjugating cells were lysed at 4.5 or 9 h post-mixing and the Twi1p complex was immunoprecipitated with anti-Twi1p antibody (IP). Coimmunoprecipitated RNA was analyzed by RT–PCR (RT+) to detect M-MDS or M-IES transcripts. RNA was also extracted from part of the lysate and analyzed by RT–PCR (Input). As a negative control, a similar experiment was performed without the reverse transcription reaction (RT−).

H3K9/K27me in ΔEMA1 cells. Wild-type (WT, top) and ΔEMA1 (bottom) conjugating cells at 9 or 12 h post-mixing were processed for immunofluorescent staining (green) using anti-H3K27me3 (A–D), anti-H3K9me2 (E–H), anti-H3K9me3 (I–L), or anti-Ema1p (M–P) Abs. DNA was stained with DAPI (purple). Arrowheads indicate developing new Macs.