Life cycle and nuclear dimorphism of Tetrahymena

(A) Under nutrient-rich conditions, vegetative Tetrahymena cells proliferate by binary fission, with the macronucleus (MAC) and micronucleus (MIC) dividing independently (a). After prolonged starvation, two cells of complementary mating types fuse to initiate conjugation, the sexual reproduction process (b). During meiotic prophase, the MICs elongate into a crescent shape, in which chromosome arms are aligned in parallel, with centromeres and telomeres anchored at opposite ends (c). The MICs then undergo meiosis (d), and one meiotic product survives and divides mitotically to produce two gametic pronuclei (e). One of the two pronuclei of each side traverses the conjugation bridge (f) and fuses with the stationary pronucleus to form a diploid zygotic nucleus (g). The zygotic nucleus undergoes two mitotic divisions (h), producing four nuclei: two differentiate into MAC anlagen (An), while the other two remain as MICs; the parental MACs are degraded (i). The conjugating pair then separates to form exconjugants (j), which resume vegetative growth when nutrients are restored (k, l). (B) The MIC is diploid, with five chromosomes per haploid genome. MIC telomeres consist of distal G4T2 (5′-GGGGTT-3′) repeats (green) and proximal G4T3 (5′-GGGGTTT-3′) repeats (purple). In contrast, the MAC is polyploid (~90 copies) and contains 181 chromosomes per haploid genome. MAC telomeres are composed exclusively of G4T2 repeats. The MAC is derived from the MIC during sexual reproduction, when chromosome breakage followed by de novo addition of G4T2 repeats generates MAC chromosomes. During this genome rearrangement process, MIC telomeres are thought to be eliminated from the developing MAC. Programmed DNA elimination of internal eliminated sequences (IESs) is not depicted.

Elimination of MIC telomeres during MAC development

Vegetative (Veg) and conjugating wild-type cells at the indicated time points (hours post-mixing, hpm) were analyzed by oligo-FISH using an oligonucleotide probe complementary to the MIC-specific G4T3 repeat (magenta). DNA was counterstained with DAPI (blue). Insets show enlarged images of the regions indicated by dotted squares. The presence of the G4T3 FISH signal was quantified in 50 cells per time point and categorized according to the number of G4T3 dots per new MAC (An) (≥6 dots, <6 dots, or none). Arrowheads with “a”, “i”, “p”, and “n” indicate the MAC, the MIC, the parental MAC and the new MAC, respectively. Scale bars: 10 µm.

Identification of MIC-limited sequences at the ends of MIC chromosomes.

(A) The MIC genome assembly (version 2021) and the MAC genome assembly (version 2025) were compared using Minimap2, and the coverage of each MIC chromosome sequence by the MAC genome sequence is shown as a histogram (blue). All MIC chromosome ends consist of 276–463 kb MIC-limited sequences (MLSs, marked by red or orange boxes) that are not represented in the MAC genome sequence. (B) In eight of the ten MIC chromosome ends (red boxes in A), a chromosome breakage sequence (CBS) was found ~20 nt distal to the boundary between MLSs (red) and MAC-destined sequences (MDSs, blue). The ends of the MDSs are directly linked to MAC telomeres (green). At the remaining MIC chromosome ends (orange boxes in A), no CBS was detected near the MLS–MDS border. (C) MIC telomeres are predicted to be separated from the MDSs by chromosome breakage at the CBS together with their associated MLSs and eliminated from the developing MAC. In parallel, the exposed MDS ends are subjected to ~20 nt resection followed by de novo addition of G4T2 telomeres.

Elimination of 4R-MLS during MAC development in wild-type cells

Vegetative (Veg) and conjugating wild-type cells at the indicated time points (hours post-mixing, hpm) were analyzed by oligo-FISH using pool of oligonucleotide probes complementary to 4R-MLS (magenta) or 4R-MDS (green). DNA was counterstained with DAPI (blue). Insets show enlarged images of the regions indicated by dotted squares. The presence or absence of the 4R-MLS FISH signal in new MAC (An) in 50 cells per time point was examined. Arrowheads with “a”, “i”, “p”, and “n” indicate the MAC, the MIC, the parental MAC and the new MAC, respectively. Scale bars: 10 µm.

Production of mutants with deletions at 4R-CBS

(A) The most distal CBS at the right arm of MIC chromosome 4 (4R-CBS), which separates MLS (red) and MDS (blue), was targeted for mutagenesis using Cas9 and a guide RNA (gRNA) complementary to the 4R-CBS locus (see B, top). Heterozygous 4R-CBS mutants were obtained and then subjected to Round I genomic exclusion to generate homozygous 4R-CBS mutants. See Materials and Methods for detailed genetic procedures. (B) The 4R-CBS locus of two mutants derived from the B2086 strain (B6-1-13 and B6-1-18) and two mutants derived from the CU428 strain (C8-1-1 and C8-1-10) was compared with that of the wild-type strain (WT) by genomic PCR followed by Sanger sequencing. All 4R-CBS mutants carried deletions (red boxes) that removed essential conserved sequences of the CBS (pink box, top).

Effect of 4R-CBS mutations on elimination of 4R-MLS

(A) Exconjugants at 30 hours post-mixing (hpm) from crosses between two wild-type strains (WT × WT), two 4R-CBS mutant strains (Mut × Mut), or a wild-type and a 4R-CBS mutant strain (WT × Mut) were analyzed by oligo-FISH using a pool of oligonucleotide probes complementary to 4R-MLS (magenta) and 4R-MDS (green). DNA was counterstained with DAPI (blue). Insets show enlarged views of regions marked by dotted squares. The presence or absence of the 4R-MLS FISH signal in the developing MAC (An) was examined in 50 cells per cross. Arrowheads with “i” and “n” indicate the MIC and the new MAC, respectively. Scale bars: 10 µm. (B) Exconjugants from a cross between a wild-type strain and a 4R-CBS mutant strain (WT × Mut) at the indicated time points were analyzed as in (A).

Effect of 4R-CBS mutations on the behavior of 4R-MDS

Exconjugants from a cross between two wild-type strains (WT × WT) and two independent crosses between a wild-type and a 4R-CBS mutant strain (WT × Mut, Cross 1 and Cross 2) were analyzed by oligo-FISH using a pool of oligonucleotide probes complementary to 4R-MDS. The number of 4R-MDS foci per developing MAC (An) at 12 hpm (n = 204), 13.5 hpm (n = 140), and 15 hpm (n = 202) was counted, and the mean ± standard error of the mean (SEM) was plotted. Statistical significance between WT × WT and WT × Mut Cross 1 (red) or Cross 2 (magenta) was assessed by Student’s t-test (ns: p > 0.05; **p ≤ 0.01; ***p ≤ 0.001).

Effect of 4R-CBS mutations on progeny viability, DNA elimination, and heterochromatin formation

(A) The indicated sets of strains were crossed, and the presence (+) or absence (−) of viable sexual progeny was examined. See Materials and Methods for details. (B, C) Exconjugants at 32 hours post-mixing (hpm) (B) or conjugating pairs at 9 hpm (C) from a cross between two wild-type strains (WT × WT) and two independent crosses between a wild-type and a 4R-CBS mutant strain (WT × Mut, Cross 1 and Cross 2) were analyzed. FISH was performed using probes complementary to the MIC-specific Tlr1 element (magenta) (B), and immunofluorescence staining was performed using an antibody against di- and tri-methylated histone H3 at lysine 9 (H3K9me, green) (C). DNA was counterstained with DAPI (blue). The presence or absence of the Tlr1 FISH signal (B) or H3K9me immunostaining signal (C) in the developing MAC (An) was examined in 50 cells per cross. Arrowheads with “i”, “p”, and “n” indicate the MIC, the parental MAC and the new MAC, respectively. Scale bars: 10 µm.

Elimination of Tlr1 element during MAC development

Conjugating wild-type cells at the indicated time points (hours post-mixing, hpm) were analyzed by FISH using probes complementary to the MIC-specific Tlr1 element (magenta). DNA was counterstained with DAPI (blue). The Tlr1 FISH signal in the developing MAC (An) was examined in 50 cells per time point and classified according to staining pattern: Homogeneous, signal distributed throughout the new MACs; DE-body, signal localized to peripheral foci (DNA elimination bodies); or No staining, no detectable signal. Scale bars: 10 µm.

Elimination of 3L-MLS during MAC development in wild-type cells

Conjugating wild-type cells at the indicated time points (hours post-mixing, hpm) were analyzed by oligo-FISH using pool of oligonucleotide probes complementary to 3L-MLS (magenta). DNA was counterstained with DAPI (blue). Insets show enlarged images of the regions indicated by dotted squares. The presence or absence of the 3L-MLS FISH signal in new MAC (An) in 50 cells per time point was examined. Scale bars: 10 µm.