The Tetrahymena p80/p95 Complex Is Required for Proper Telomere Length Maintenance and Micronuclear Genome Stability  Michael C. Miller, Kathleen Collins  Molecular Cell  Volume 6, Issue 4, Pages 827-837 (October 2000) DOI: 10.1016/S1097-2765(05)00078-X

Figure 1 Generation of Macronuclear Knockouts and Knockdowns (A) A wild-type (WT) Tetrahymena cell is transformed with a construct designed to disrupt the coding sequence of a telomerase gene with a cassette that confers resistance to the drug paromomycin. This construct integrates by homologous recombination in one or a few of about 45 chromosomes carrying the targeted gene (“Transformed”), converting it from wild type (black bars) to disrupted (white bar). Serial passaging into increasing concentrations of paromomycin selects for cells that have accumulated more copies of the recombinant chromosome (“Partial Assortment”). When passaged at the highest tolerated concentration of paromomycin (“Maximum Assortment”), cells will be in one of two states. If the targeted gene is not essential for vegetative growth, all of the wild-type chromosomes will have been replaced with recombinant chromosomes. If the gene is essential, some number of wild-type chromosomes will remain in all viable cells. (B) Total genomic DNA was collected from wild-type (WT, lanes 1 and 5) or transformed cells (T, lanes 2–4 and 6–8). Transformed cells were maximally phenotypically assorted in paromomycin and then grown for 0 (lanes 2 and 6), 20 (lanes 3 and 7), or 100 (lanes 4 and 8) doublings without paromomycin. DNA was digested, resolved, and hybridized with probes for sequence adjacent to the site of integration of the selectable marker at each of the targeted loci. TERT is encoded by TRT, telomerase RNA by TER, p80 by TPA, and p95 by TPB. H4-I is one of two genes encoding histone H4. (C) Total cellular protein from cells grown vegetatively to mid-log phase was resolved by SDS–PAGE and probed for p95 (top) or p80 (bottom) by immunoblotting. Relative sample loading is indicated below each lane, with 1× representing 104 cells. Molecular Cell 2000 6, 827-837DOI: (10.1016/S1097-2765(05)00078-X)

Figure 2 Monster Phenotypes in δRNA and δTERT Lines Wild-type (A and B), δRNA (C and D), and δTERT (E and F) cells were fixed, stained, and examined by Nomarski optics (A,C, and E) and by DAPI staining for DNA (B,D, and F) as previously described (Yu et al. 1990). The scale bar = 50 μM. Molecular Cell 2000 6, 827-837DOI: (10.1016/S1097-2765(05)00078-X)

Figure 3 Telomerase Subunit Interactions in Δ80 and Δ95 Lines (A) An equal amount of total protein from S-100 extracts of the wild-type or knockout cells indicated was resolved by SDS–PAGE and then probed for p95 (top) or p80 (middle) by immunoblotting. A nonspecific protein recognized on the p80 immunoblot is shown to confirm equal loading (bottom), which was also evident by Ponceau S staining of the blot before antibody application (not shown). (B) Total RNA from vegetatively grown cells of the indicated lines was resolved by denaturing gel electrophoresis then probed for telomerase RNA (top) or 5S RNA (bottom) by hybridization with a complementary radiolabeled oligonucleotide. Each set of lanes represents serial 5-fold dilutions of the same amount of total RNA. Molecular Cell 2000 6, 827-837DOI: (10.1016/S1097-2765(05)00078-X)

Figure 4 Macronuclear Telomere Length in Knockout and Knockdown Lines Macronuclear telomeres in HindIII-digested genomic DNA were resolved by denaturing gel electrophoresis and then detected by hybridization with an oligonucleotide specific for the high copy number palindromic rDNA chromosome. Mean fragment length (telomeric repeats + subtelomeric DNA) and mean telomere length (telomeric repeats only) were determined as described in Experimental Procedures. Relative telomere length was calculated by normalizing the mean telomere length of each strain to mean telomere length of the wild-type strain. Molecular Cell 2000 6, 827-837DOI: (10.1016/S1097-2765(05)00078-X)

Figure 5 Telomerase Activity in Knockout Lines (A) Total protein from equal numbers of cells grown vegetatively to mid-log phase was resolved by SDS–PAGE and probed for p80 and p95 by immunoblotting. (B) Telomerase RNA levels in extracts assayed in (C)–(E). (C) Telomerase activity was assayed with 400 nM (T2G4)3 at two, 3-fold different concentrations of each extract. (D) Telomerase activity was assayed with 5 or 20 μM of the nontelomeric primer CTEX3 (Gandhi and Collins 1998). (E) Telomerase activity assays with the telomeric primer (T2G4)3 begun in the presence of [α-32P]dGTP and then stopped after 2.5 min (lanes 1 and 6) or chased for an additional 57.5 min in reaction buffer containing unlabeled dGTP and either no additional primer (lanes 2 and 7) or 1 μM primer (lanes 4 and 9). In the control reactions (lanes 3, 5, 8, and 10), chase mix was added at time zero and reactions were stopped after 60 min. Chased products did not resolve into discrete bands. Molecular Cell 2000 6, 827-837DOI: (10.1016/S1097-2765(05)00078-X)

Figure 6 Micronuclear Telomere Length in Wild-Type, Δ95, and Δ80Δ95 Lines Micronuclear telomeres in MseI-digested genomic DNA were resolved by agarose gel electrophoresis and then detected by hybridization with an oligonucleotide recognizing the micronuclear-specific, centromere-proximal T3G4/C4A3 repeats. Length was calculated as for macronuclear telomeres, except that the entire restriction fragment length including the T3G4/C4A3 repeats is reported. Sharp bands in all lanes represent cross-hybridizing nontelomeric DNA fragments. (A) Micronuclear telomeres were examined in the indicated CU428 lines generated at different times. DNA from the Δ80Δ95 line is underloaded relative to the other samples. (B) Micronuclear telomeres were examined in simultaneously generated Δ80Δ95 lines in CU427 and CU428 backgrounds. Molecular Cell 2000 6, 827-837DOI: (10.1016/S1097-2765(05)00078-X)

Figure 7 A Mating Defect in Cells Lacking p80/p95 Conferred by Damage in the Micronucleus (A) Diagram of a Δ80Δ95 × Δ80Δ95 cross generating true progeny. Parental micronuclei carry unexpressed genes encoding cycloheximide resistance (chx1-1) or 6-methylpurine resistance (mpr1-1). Parental macronuclei carry the NeoR gene disrupting the genes encoding p80 and p95. Cells are resistant to paromomycin and sensitive to cycloheximide and 6-methylpurine (cy-s, mp-s, pm-r). Upon true mating, paromomycin resistance is lost while cycloheximide and 6-methylpurine resistance are gained (cy-r, mp-r, pm-s). (B) Quantitation of mating efficiency. Mating pairs were isolated from the crosses indicated; Δ80Δ95pop indicates a culture approximately 100 generations following isolation of a single cell as the final step of phenotypic assortment, Δ80Δ95clone indicates a culture that arose from a single cell isolated from the population culture, and Δ80Δ95new mic indicates a line expanded from a clone that had its damaged micronucleus replaced with a wild-type micronucleus by genomic exclusion. Approximately ten doublings of vegetative growth after mating, progeny were tested for resistance or sensitivity to cycloheximide, 6-methylpurine, and paromomycin. cy-r, mp-r, pm-s clones were scored as truly mated (black); all other viable progeny were pooled into one class (white). These included progeny that had undergone genomic exclusion, uniparental cytogamy, or true mating in which parents contributed incomplete genomes. Viability of each Δ80Δ95 × Δ80Δ95 cross was normalized to the viability of an age-matched WT × WT cross. (C) Decreased and variable micronuclear DNA content in Δ80Δ95 populations and clones. Nuclei from wild-type cells (WT, solid), a mixed population of Δ80Δ95(CU427) cells (Δ80Δ95pop, dashed), or clone derived from the Δ80Δ95(CU428) knockout line (Δ80Δ95clone, dotted) were analyzed for DNA content by flow cytometry. Fluorescence from micronuclei and macronuclei is indicated. Molecular Cell 2000 6, 827-837DOI: (10.1016/S1097-2765(05)00078-X)