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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01078

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Suppression of a mitotic mutant by tRNA-Ala anticodon mutations that produce a dominant defect in late mitosis

Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University. Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8501, Japan

View larger version (41K): [in a new window]   Fig. 1. Phenotypes of cs scn1 and scn2 mutants. (A) Fission yeast scn1 and scn2 are extragenic suppressors for ts cut9-665 that causes the assembly defect in APC/C (Samejima and Yanagida, 1994; Yamada et al., 1997). Single cut9 mutant fails to form colonies at 33°C, whereas double mutants cut9 scn1 and cut9 scn2 form colonies at 33°C. Single scn1 and scn2 mutants are unable to form colonies at 22°C. (B) Wild-type, scn1 and scn2 grown at 33°C were transferred to 20°C and cultured for 50 hours in the complete YPD medium. The cell number of wild-type (WT), scn1 and scn2 mutants was scored. (C) The cell length of wild-type (hatched columns) and scn1 mutant (red columns) was measured after 0 and 24 hours culture at 20°C. The average cell length of wild-type and scn1 mutant is indicated. (D) Light micrographs of wild-type and scn1 mutant cells cultured at 20°C for 9 hours. Wild-type and scn1 mutant cells were cultured at 20°C, fixed with glutaraldehyde and stained with DAPI. The arrows indicate the displaced divided nuclei abundantly present in scn1 and scn2 mutant cells. Bar, 10 µm.

View larger version (35K): [in a new window]   Fig. 2. Single scn1 mutant is defective in spindle elongation, nuclear positioning and cell division, producing frequent late mitotic phenotypes at 20°C. (A) Wild-type (WT) and scn1-17 grown at 33°C were transferred to 20°C in the YPD medium. Anti-tubulin (red), anti-Sad1 (green) and DAPI (blue) staining were done for wild-type and scn1 mutant cells after 9 hours and 12 hours at 20°C. Late anaphase cells, revealing separated chromatids and a spindle that was not fully elongated, were marked as A. The nuclear displacement indicated as B was often seen in scn1 mutant cells. Cells lacking the nuclei were also seen (an example indicated as scn1-17 in right panel). Bar, 10 µm. (B) The frequency of late anaphase cells in the wild-type and scn1-17 mutant after the transfer to 20°C for 0-12 hours. Cells in late anaphase were significantly accumulated after 9 hours in scn1-17. (C) The frequencies of wild-type (upper) and scn1 mutant (lower) cells showing normal positioning of the separated daughter nuclei (open rectangles), abnormally displaced daughter nuclei (filled triangles) and no nuclei (crosses) are shown.

View larger version (30K): [in a new window]   Fig. 3. The loss of viability in scn1-17 coincides with aberrant mitosis. (A) The wild-type and scn1-17 cells were first arrested at the G1 phase under the nitrogen source starvation at 33°C, and then shifted to the complete YPD medium at 20°C. The DNA contents of wild-typ e and scn1-17 were determined by FACS analysis. In wild-type, the S phase (S) occurred from 6 to 12 hours, whereas, in scn1-17, the progression of S phase was delayed by 1.5 hours (6-13.5 hours). 1C indicates the DNA content of a haploid cell in G1 that has unreplicated DNA, whereas 2C indicates those of a G2 cell that has already replicated DNA once. (B) Upper panel: the cell viability (Viability%) and septation index (SI%) were measured after the release from the G1 arrest at various time points in wild-type and scn1 mutant cells. The scn1 mutant cells lost the viability during the passage through the first mitosis, with the concomitant increase of missegregation. Lower panel: the frequency of mitotic phenotype in scn1-17 was determined. Frequency (%) of chromosome missegregation in scn1 (displaced, separated daughter nuclei) was determined (lower panel).

View larger version (46K): [in a new window]   Fig. 4. Anaphase spindle elongation was slow and not complete in scn1-17 mutant. (A) The spindle index of wild-type and scn1-17 was measured after transfer to 20°C. The short spindle (the metaphase spindle length 2.5 µm) did not accumulate in scn1-17. (B) Cdc13 (mitotic cyclin in fission yeast) is degraded in aberrant anaphase in scn1. The scn1 mutant carrying chromosomally integrated cdc13-GFP gene was cultured at 20°C for 9 hours in the liquid EMM2, harvested and stained with Hoechst H33342 (DNA). The Cdc13-GFP signals (strong in the nucleus) faded in all of the anaphase cells (arrow) examined in scn1-17, as well as in wild-type cells. Bars, 10 µm. (C) The wild-type and scn1-17 mutant cells carrying plasmid pSAD1-GFP (Sad1 is a SPB protein) were observed for the spindle dynamics in living cells. Cells were cultured at 20°C for 9 hours. The white number in each panel shows the time (minutes) after starting the observations using a confocal micr oscope. Full anaphase spindle extension was complete within 40 minutes in wild-type cells, whereas spindle elongation was slow and incomplete after 96 minutes in scn1-17 mutant cells. Furthermore, the nuclear envelope in the middle appeared to remain in late anaphase, suggesting that nuclear division was not finished. Bars, 10 µm. (D) Distances between the SPBs were measured in living cells. In the scn1 mutant cells, the rate of anaphase spindle elongation was strikingly low and the SPBs did not reach the opposite poles of the cells, even after more than 100 minutes.

View larger version (51K): [in a new window]   Fig. 5. scn1 and scn2 are both Ala-tRNA anticodon mutants. (A) The genomic DNAs were purified from wild-type, scn2-7 and scn2-27 cells. The 2 kb long genomic DNA fragments corresponding to the 2425-4520 region of the chromosome I cosmid c4C5 were amplified by PCR, followed by DNA sequencing. Amplified DNAs from the mutant strains scn2-7 and scn2-27 showed a single mutation (indicated in red) in alanine-tRNA gene (SpaTRNAala.02). Otherwise the sequences were identical to that obtained from the wild-type. (B) The predicted cloverleaf structure of the tRNA-Ala (UGC) in wild-type and scn2. The mutated scn2 tRNA is presumed to recognize the ACA codon in mRNA that encodes threonine. (C) The plasmid containing the mutant tRNA gene of scn2 conferred cold-sensitivity on wild-type (WT) cells and suppressed the temperature sensitivity of cut9-665. Wild-type and cut9-665 (cut9) carrying plasmid of the wild-type tRNA gene (pTRNAala.02) and plasmid of scn2 mutant tRNA gene (pTRNAala.02(UGC>UGU)) were incubated on the minimal EMM2 plate at 22, 26, 33 and 36°C. (D) Wild-type cells carrying multicopy plasmid containing the mutant Ala-tRNA(UGU) gene showed mitotic phenotypes sim ilar to those of scn1 and scn2 mutants. Wild-type cells carrying pTRNAala.02 or pTRNAala.02(UGC>UGU) were cultured in the EMM2 medium at 20°C for 9 hours and fixed with glutaraldehyde. DNA was stained with DAPI. The phenotype of scn1-17 was also shown. Brightly stained cells were dead and accumulated the dye. Bar, 10 µm. (E) The scn1 mutant contained a substitution mutation in the second copy of alanine-tRNA gene, but not in the scn3+ gene. The genomic DNA of wild-type and scn1-17 was purified, and DNA sequencing of the region containing the second tRNA gene (SpaTRNAala.03) was done. The SpaTRNAala.03 near to the scn3+ gene was mutated in scn1-17 in the same position in anticodon as in scn2.

View larger version (79K): [in a new window]   Fig. 6. Drug sensitivities of scn1 and scn2 tRNA mutants, and high dosage suppression of scn mutants by Scn3 and Thr-tRNA. (A) Mutants scn1 and scn2 were hypersensitive to cycloheximide, a protein synthesis inhibitor. Wild-type, mis3-224 (Kondoh et al., 2000), scn1-17, scn2-7 and scn3+-disrupted cells (Samejima and Yanagida, 1994) were spotted on the YPD plate containing respective concentrations of cycloheximide. (B) Mutants scn1 and scn2 are hypersensitive to TBZ, a microtubule destabilizing drug at 26°C, the semi-permissive temperature. Wild-type, ß-tubulin mutant nda3-KM311 (Toda et al., 1983), scn1-17 and scn2-7 were spotted on the YPD plate containing respective concentrations of TBZ. (C) The scn3+ gene is a multicopy suppressor for scn1 and scn2 mutants. scn1-17 and scn2-7 mutants were transformed with plasmid containing the scn3+ gene under the inducible nmt1-promoter (pREP1-scn3+). Transformants were plated on the EMM2 containing thiamine (promoter, repressible condition). (D) Plasmid containing the Thr-tRNA(UGU) gene (pTRNAthr.06 and pTRNAthr.10) could suppress the cold-sensitivity of scn1 and scn2 mutant cells.

View larger version (46K): [in a new window]   Fig. 7. The delay of spindle elongation in scn1-17 was not dependent on the spindle checkpoint. (A) The double mutant scn1-17 mad2 cells were cultured at 20°C for 9 hours in the YPD medium and cells were fixed for staining by antibodies against tubulin (red) and Sa d1 (green). DAPI (blue) staining was done for DNA. The late anaphase cells were designated as A. Late anaphase cells were observed in scn1 mad2 as frequent as in the scn1 single mutant cells. Bar, 10 µm. (B) The pole-to-pole distance in mitotic cells was measured in the wild-type, scn1 and scn1 mad2 cells that were cultured at 20°C for 9 hours in YPD. Cells were stained with anti-Sad1 (SPB) and anti-TAT1 (tubulin). Over 100 mitotic cells of each strain were analyzed. The filled arrowheads indicate the size of the wild-type spindle at metaphase. The solid bar indicates the sizes of anaphase spindle that were abundantly seen in scn1 and scn1 mad2 cells. The cells showing anaphase spindle were accumulated in scn1 mad2 as much as in scn1 single mutant cells.