Article Figures & Data
Figures
- Fig. 1.
DIE5 is a developmentally regulated gene encoding a nuclear protein in Paramecium. (A) Northern blot of total RNA (20 μg per lane) from Paramecium probed with ParameciumDIE5a. Ethidium bromide staining of rRNA was used as a loading control. (B) Cellular localization of GFP-Die5a expressed from an extrachromosomal DNA driven by the DIE5a promoter. Fluorescent images are projections of optical sections obtained by confocal microscopy. The GFP fluorescence exclusively localized to the new macronuclei, which show weaker propidium iodide staining (DNA) than the old macronuclear fragments. The image corresponds to a cell approximately 14 h after the start of mating. The bar corresponds to 20 μm.
- Fig. 2.
Silencing of DIE5 disrupts nuclear events during conjugation. (A) Progeny from conjugation treated with RNAi. Survival (%) and phenotype of the marker genes are shown. Both parental strains for conjugation were homozygous for Mendelian recessive mutant alleles of different marker genes (pwA and pwB; required for ciliary reversal); thus, successful conjugation should produce the wild-type phenotype of backward swimming, while failure in either nuclear exchange, fertilization, or formation of the new macronucleus should result in the mutant phenotype, i.e., no backward swimming upon stimulation. (B) Representative DAPI-stained Paramecium exconjugants with normal and defective cytological phenotypes observed after RNAi treatments. Each picture contains a single cell with or without the new macronucleus (arrowheads) and old macronuclear fragments. Phenotypic classes were assigned to white, gray, or black, as indicated below the pictures and plotted in the graphs in panel C. mac, macronucleus.
- Fig. 3.
Silencing of DIE5 inhibits excision of IESs. (A) A partial map of the micronuclear version of the A-51 allele showing locations of IESs (boxes), SspI recognition sites (arrowheads), and sizes of fragments generated by SspI digestion. Positions of expected PCR products (pp1 to pp3) and probes for Southern hybridization are also indicated. Arrows show positions of the primers relative to IESs not drawn to scale. (B) Whole-cell semiquantitative PCRs of RNAi-treated exconjugants, using one primer in the macronuclear sequence and the other primer inside the IESs. Each lane represents whole-cell PCR products taken at 2-h intervals from 6 to 22 h after induction of conjugation. The predicted PCR products correspond to pp1 to pp3 in panel A. Due to excision of smaller IESs during rearrangement, two bands are expected for each primer set. (C) SspI-digested total genomic Southern blots of RNAi-treated exautogamous cells probed with either a HincII-PstI fragment or IES4578, as indicated in panel A. Total DNA (∼10 μg) was isolated from an exautogamous cell culture when about 50% of control cells (RNAi using empty vector) had undergone the first cell division. Most IESs in the micronuclear version of the A-51 allele contain SspI sites (single arrow heads in panel A), while only one site is present in the macronuclear-destined sequence of the A-51 allele (the double arrow head in panel A). Thus, for probe HincII-PstI, 1.3- and 2.0-kb fragments are expected for unprocessed DNA, while a 5.7-kb fragment is expected for the processed DNA, including abundant old macronuclear DNA in exconjugants. Probe IES4578 contains only the IES sequence and detects 0.5-kb fragments if the IES is present at high levels in exautogamous cells.
- Fig. 4.
Tetrahymena homologue (TtDIE5) is developmentally expressed. (A) Pairwise alignment of Paramecium Die5 protein from paralog a (PtDIE5a) and Tetrahymena Die5p sequences. The boxes around three amino acids indicate differences between the two Paramecium sequences (the differences are C, D, D, respectively). The underlined regions indicate potential nuclear localization signals in the Paramecium sequence. (B) Northern blot analysis of total RNA (20 μg per lane) extracted from growing (log), starved, or conjugating cells (between 2 and 25 h after mixing populations of compatible mating types). Ethidium bromide staining of rRNA was used as a loading control.
- Fig. 5.
TtDIE5 zygotic expression is essential to complete conjugation. (A) Diagram of the gene disruption construct showing replacement of the coding sequence with the neo3 selectable cassette (47). Restriction enzyme sites (BstBI) and the region corresponding to the radiolabeled probe fragment used for Southern blot analysis are indicated. WT, wild type; MTT1pr, metallothionein gene 1 promoter. (B) Southern blot hybridization of DNA isolated from the wild type (lane 1), DIE5 micronuclear-knockout lines (lanes 2 to 5), and the DIE5 complete knockout line (lane 6) using the probe shown in panel A. Longer exposure of the blot reveals the 3.5-kb band in DIE5 micronuclear-knockout samples corresponding to the two copies of DIE5 disrupted by the neo3 cassette in their micronuclei. (C) RT-PCR for expression of DIE5 transcripts in conjugating wild-type (wt) and DIE5 complete knockout (Δ) cells at indicated time points. The bottom panel shows control RT-PCR with HHP1 primers. Both DIE5 and HHP1 primers span an intron of their respective genes. g, Tetrahymena genomic DNA used as a control for amplification. (D) Fluorescent images of representative DAPI-stained wild-type (WT), micronuclear-knockout (ΔDIE5), and ΔPDD1 strains at 9 h, 14 h, and 33 h postmixing. For each mating strain, the percentage of cells exhibiting their respective arrest phenotype at 33 h is indicated on the right. Asterisks, arrows, and arrowheads indicate old/parental macronuclei, new macronuclei, and the micronuclei, respectively. (E) The nuclear envelope remains intact in arrested ΔDIE5 micronuclear-knockout cells (ΔDIE5). Postconjugative ΔDIE5mic cells were fixed with 2% paraformaldehyde and stained with αNopp52 antibody and DAPI. New macronuclei and micronuclei are indicated as described for panel D.
- Fig. 6.
Cellular localization of GFP-Die5p in conjugating Tetrahymena cells at 6 h, 8 h, and 12 h. The cells were fixed with 2% paraformaldehyde and counterstained with DAPI. Asterisks, arrows, and arrowheads indicate old/parental macronuclei, new macronuclei, and the micronuclei, respectively.
- Fig. 7.
Germ line TtDIE5 is required for Tetrahymena-programmed DNA rearrangement. Total genomic DNA was isolated from starved wild-type (WT) and DIE5 micronuclear-knockout strains (ΔDIE5), as well as from cells 16 h after crosses of DIE5 micronuclear knockouts to the wild type (WT × ΔDIE5) and two DIE5 micronuclear knockouts (ΔDIE5 × ΔDIE5). The DNA was used for PCR-based IES excision assays (A and B) or Southern blot analyses for chromosome breakage (C and D). (A) Schematic of PCR-based IES excision assay strategy. Arrows denote forward and reverse primers used to amplify across the M element. Alternative rearrangement products resulting from deletion of 0.6-kbp (Δ0.6) or 0.9-kbp (Δ0.9) are shown. (B) M-element excision PCR. Arrow indicates new IES excision. (C) Diagram shows the macronuclear chromosomal scaffold surrounding the LIA1 gene, which lies within 2.2 kbp of a chromosomal-breakage sequence (CBS) (white star). Relevant EcoRI (RI) restriction sites used for the Southern blot analysis are shown. The probe spans the central EcoRI site and detects a 7.8-kbp fragment common to both nuclei as well as to either the 10.5-kbp micronucleus-specific fragment or a 2.5- to 2.6-kbp macronucleus-specific fragment (2.2 kbp of unique sequence plus 300 to 400 bp of telomeric DNA). Tel, telomere. (D) Southern blot analysis to assess chromosome breakage. Arrow indicates the product of de novo breakage.
- Fig. 8.
Germ line knockout of TtDIE5 does not inhibit critical events leading to IES excision. (A) H3K27me3 and H3K9me2 histone mark deposition is unaffected in TtDIE5 germ line knockouts. Nine-hour-conjugating wild type (WT) and micronuclear-knockout (ΔDIE5) cells were fixed with Schaudinn's fixative and stained with either H3K27me3 or H3K9me2 antibodies at 9 h into conjugation. The cells were counter stained with DAPI. (B) Formation of PDD1 foci is not disrupted in ΔDIE5mic cells. Conjugating wild-type and DIE5 micronuclear-knockout cells (ΔDIE5) expressing an inducible PDD1-YFP transgene were fixed at 14 h with 2% paraformaldehyde and counter stained with DAPI. Asterisks, arrows, and arrowheads indicate old/parental macronuclei, new macronuclei, and the micronuclei, respectively.
