Cold-sensitive nuclear division arrest mutants of the fission yeast Schizosaccharomyces pombe

Thirteen recessive cold sensitive nuclear division arrest mutants were isolated from the fission yeast Schizosaccharomyces pombe. Twelve unlinked genes were defined; six in chromosome I, three in chromosome II and two in chromosome III. The map positions of three nuclear division arrest genes (nda1, nda2 and nda3) in chromosome II were determined precisely. Together with the previously obtained temperature-sensitive cell division cycle mutations, at least 20 genes appear to control the nuclear division of the fission yeast. Physiological studies indicated that most cold sensitive nda mutants incubated previously at 22 degrees C proceeded with a synchronously normal cell-cycle after temperature shift-up. The morphology of the nuclei and nuclear chromatin region was studied by the 4',6-diamidino-2-phenylindole staining method and by electron microscopy. Each mutant exhibited characteristic nuclear morphology at 22 degrees C, showing the specific blockages. The nda genes seem to control a pathway of structural alterations in the nuclear chromatin region with the order hemisphere, condensed ellipsoid, segregating U-form and separating hemispheres. Two genes, nda2 and nda3, pleiotropically control nuclear division, nuclear location and cell shape. The terminal phenotype of nda2-KM52 is characterized by the nuclear displacement, the absence of a spindle and abnormal locations of spindle pole bodies. The cells of nda3-KM311 were aberrant in shape and contained a partially separated chromatin region with a long spindle. Together with the results of the accompanying paper, we conclude that nda2 and nda3 genes control nuclear and cytoplasmic microtubular organization.     

Effects of pressure stress on the fission yeast Schizosaccharomyces pombe cold-sensitive mutant nda3

To investigate the influence of pressure stress on the cell cycle of Schizosaccharomyces pombe, we used a cold-sensitive nda3-KM311 mutant which arrests cell division at a step similar to the mitotic prophase, proposed by Hiraoka and colleagues (Cell 39 (1984) 349-358), under the restrictive temperature, 20 degrees C. The nda3-KM311 cells were first aerobically grown at 30 degrees C, transferred to 20 degrees C for 4 h and shifted to a permissive temperature of 36 degrees C for 15 min. The cells were treated with 100-200 MPa pressure and studied by electron and fluorescence microscopy. At 100 MPa, the nuclear membrane was damaged and the matrix of mitochondria had an electron-dense area. At 150 MPa, the nuclear membrane was broken over broad areas; numerous small vacuoles had fused into large pieces. Actin patches were concentrated in the central region and actin rings were seen in the 20 degrees C-grown cells. Even at 100 MPa, specific actin distribution was lost. Although at 100 MPa, long and fine actin cables were seen all over the cells, large actin patches and the actin rings remained in the center of the cell. They changed into thick and short cables at 150 MPa and above 200 MPa they decomposed but the actin ring was visible even with faint fluorescence. Immunoelectron microscopic observation confirmed this phenomenon.     

Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family.

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.     

Isolation of a Schizosaccharomyces pombe rad21ts mutant that is aberrant in chromosome segregation,microtubule function,DNA repair and sensitive to hydroxyurea: possible involvement of Rad21 in ubiquitin-mediated proteolysis.

The fission yeast DNA repair gene rad21+ is essential for cell growth. To investigate the function essential for cell proliferation, we have isolated a temperature-sensitive mutant of the rad21+ gene. The mutant, rad21-K1, showed abnormal mitosis at the nonpermissive temperature. Some cells contained abnormal nuclear structures, such as condensed chromosomes with short spindles, or chromosomes stretched or unequally separated by elongating spindles. Other cells exhibited the displaced nucleus or a cut-like phenotype. Similar abnormalities were observed when the Rad21 protein was depleted from cells. We therefore concluded that Rad21 is essential for proper segregation of chromosomes. Moreover, the rad21-K1 mutant is sensitive not only to UV and gamma-ray irradiation but to thiabendazole and hydroxyurea, indicating that Rad21 plays important roles in microtubule function, DNA repair, and S phase function. The relation to the microtubule function was further confirmed by the fact that rad21+ genetically interacts with tubulin genes, nda2+ and nda3+. Finally, the growth of the rad21-K1 mutant was inhibited at the permissive temperature by introduction of another mutation in the cut9+ gene, coding for a component of the 20S cyclosome/anaphase promoting complex, which is involved in ubiquitin-mediated proteolysis. The results suggest that these diverse functions of Rad21 may be facilitated through ubiquitin-mediated proteolysis.     

Fission yeast genes which disrupt mitotic chromosome segregation when overexpressed.

An interference assay has been devised in Schizosaccharomyces pombe to rapidly identify and clone genes involved in chromosome segregation. Random S.pombe cDNAs were overexpressed from an inducible promoter in a strain carrying an additional, non-essential minichromosome. Overexpression of cDNAs derived from four genes, two known (nda3+and ubc4+, encoding beta-tubulin and a ubiquitin conjugating enzyme, respectively) and two unknown, named mlo2+ and mlo3+ (missegregation & lethal when over expressed) caused phenotypes consistent with a failure to segregate chromosomes. Full overexpression of all four cDNAs was lethal. Cells overexpressing nda3+ and ubc4+ cDNAs arrested with condensed unsegregated chromosomes and cells overexpressing mlo2+ displayed an asymmetric distribution of nuclear chromatin. Sublethal levels of overexpression of nda3+, ubc4+ and mlo2+ cDNAs caused elevated rates of minichromosome loss. A third cDNA mlo3+, displayed no increase in the frequency of minichromosome loss at sublethal levels of overexpression but full overexpression caused a complete failure to segregate chromosomes. Our results confirm the assumption that beta-tubulin overexpression is lethal in S.pombe, implicate ubc4+ in the control of metaphase-anaphase transition in fission yeast and finally identify two new genes, mlo2+and mlo3+, likely to play an important role for chromosome transmission fidelity in mitosis.     

Isolation and characterization of mutants supersensitive to the spindle poison, isopropyl N-3-chlorophenyl carbamate (CIPC) in the fission yeast Schizosaccharomyces pombe.

Mutants supersensitive to the spindle poison, Isopropyl N-3-chlorophenyl carbamate (CIPC) of the fission yeast Schizosaccharomyces pombe were isolated and characterized genetically. Fourteen different recessive loci were assigned for the mutation (donated as cps1 to cps14) and two, cps1 and cps3, were mapped precisely on the chromosomes. Nine mutant strains were also supersensitive to phenothiazine derivatives, inhibitors of calcium-binding protein calmodulin. Four of nine strains were incapable of growing in the presence of 10 microM calcium ionophore A23187, at which the drug had no effect on cell growth in other strains. Fluorescence microscopy using the DAPI and Calcofluor staining methods showed two strains out of four to be defective in normal cell division; most stationary-phase cells of the cps6 mutant were seen to be bi- or tetra-nucleate, being partitioned with one or three septa, respectively. In the other mutant (cps8), enlarged cells were unequally partitioned with multisepta, and each compartment contained several daughter nuclei. The septa appeared aberrant in position within the cell, and situated diagonally but not vertically along the long cell axis.     

DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe

We show that DNA topoisomerase II (topo II) is continuously required for mitotic chromosome changes in Schizosaccharomyces pombe. We constructed cold-sensitive (cs) or temperature-sensitive (ts) strains mutated in the genes coding for topo II (top2) and beta-tubulin (nda3). The ATP-dependent activity of the top2cs gene product is cs in vitro. The cloned top2cs gene sequence predicts an amino acid substitution. A cs top2-cs nda3 double mutant at 20 degrees C shows long, entangled chromosomes, which condense and separate upon the shift to permissive temperatures. If spindle formation is prevented at permissive temperatures, the chromosomes condense but do not separate. Thus topo II is required for final chromosome condensation; moreover, pulse-shift experiments show that topo II is required for chromatid disjuction. Experiments with ts top2-cs nda3 cells show that topo II is also required for chromosome separation in anaphase: inactivation of topo II and activation of beta-tubulin allow normal spindle formation but result in "streaked" chromosomes.     

Genetic analysis of resistant mutants to antimitotic benzimidazole compounds in Schizosaccharomyces pombe

Summary Mutants resistant to the antimitotic compounds thiabendazole and methyl-2-benzimidazolecarbamate were isolated and analyzed genetically in the fission yeast, Schizosaccharomyces pombe. They comprised three groups in terms of genetic linkage. Mutants in one linkage group (ben1) differed phenotypically from those in the other two (ben2 and ben3). The former were resistant to the compounds at any physiological temperature tested, whereas the latter exhibited temperature dependent resistance. Through tetrad analysis, ben1 was mapped at the rightmost part of chromosome II, and ben2 was mapped near the centromere of the same chromosome. Haploidization experiments revealed the location of ben3 on chromosome II. By analogy with Aspergillus nidulans, it is suggested that one of these ben genes may code for tubulin.     

Fission yeast Pot1 and RecQ helicase are required for efficient chromosome segregation

Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.     

But1 and But2 proteins bind to Uba3, a catalytic subunit of E1 for neddylation, in fission yeast

NEDD8/Rub1 is the most homologous protein to ubiquitin among the ubiquitin-like proteins, and it is covalently linked to target proteins via the C-terminal glycine residue in a manner analogous to ubiquitylation. However, the mechanism(s) involved in the regulation of the NEDD8 ligation pathway remains elusive. Using the two-hybrid system, we isolated novel genes from the Schizosaccharomyces pombe cDNA library whose products bind to Uba3, which is a catalytic protein for E1-like activity of the NEDD8 pathway. We designated these genes but1(+) and but2(+) (for proteins that bind to Uba three). But1 is a nuclear protein and its overexpression caused cell elongation, which is a common phenotype of the NEDD8 pathway defective mutant in S. pombe. Furthermore, overexpression of but1(+) in ned8-temperature sensitive mutant had a deleterious effect even under permissive temperatures. Our results suggest that But1 may have an inhibitory role in the NEDD8 pathway.     

Cold-sensitive and caffeine-supersensitive mutants of the Schizosaccharomyces pombe dis genes implicated in sister chromatid separation during mitosis

We isolated novel classes of Schizosaccharomyces pombe cold-sensitive dis mutants that block mitotic chromosome separation (nine mapped in the dis1 gene and one each in the dis2 and dis3 genes). Defective phenotype at restrictive temperature is similar among the mutants; the chromosomes condense and anomalously move to the cell ends in the absence of their disjoining so that they are unequally distributed at the two cell ends. Synchronous culture analyses indicate that the cells can enter into mitosis at normal timing but become lethal during mitosis. In comparison with the wild-type mitosis, defects are found in the early spindle structure, the mitotic chromosome structure, the poleward chromosome movement by the spindle elongation and the telophase spindle degradation. The dis mutants lose at permissive temperature an artificial minichromosome at higher rates than occur in the wild type. We found that all the dis mutants isolated are supersensitive to caffeine at permissive temperature. Furthermore, the mutant cells in the presence of caffeine produce a phenotype similar to that obtained at restrictive temperature. We suggest that the dis genes are required for the sister chromatid separation at the time of mitosis and that caffeine might affect the dis gene expression. We cloned, in addition to the dis2+ and dis3+ genes, multicopy extragenic suppressor sequences which complement dis1 and dis2 mutations. A complex regulatory system may exist for the execution of the dis+ gene functions.     

Characterization of a Schizosaccharomyces pombe strain deleted for a sequence homologue of the human damaged DNA binding 1 (DDB1) gene

Human damaged DNA-binding protein (DDB) is a heterodimer of p48/DDB2 and p127/DDB1 subunits. Mutations in DDB2 are responsible for Xeroderma Pigmentosum group E, but no mutants of mammalian DDB1 have been described. To study DDB1, the Schizosaccharomyces pombe DDB1 sequence homologue (ddb1(+)) was cloned, and a ddb1 deletion strain was constructed. The gene is not essential; however, mutant cells showed a 37% impairment in colony-forming ability, an elongated phenotype, and abnormal nuclei. The ddb1Delta strain was sensitive to UV irradiation, X-rays, methylmethane sulfonate, and thiabendazole, and these sensitivities were compared with those of the well characterized rad13Delta, rhp51Delta, and cds1Delta mutant strains. Ddb1p showed nuclear and nucleolar localization, and the aberrant nuclear structures observed in the ddb1Delta strain suggest a role for Ddb1p in chromosome segregation.     

Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation.

The Schizosaccharomyces pombe sts1+ gene, identified by supersensitive mutations to a protein kinase inhibitor, staurosporine, was isolated by complementation by the use of a fission yeast genomic library. Nucleotide sequencing shows that the sts1+ gene encodes a 453 amino acid putative membrane-associated protein that is significantly similar (26% identity) to the chicken lamin B receptor. It is also highly related (53% identity) to a budding yeast ORF, YGL022. These three proteins contain a similar hydrophobicity pattern consisting of eight or nine putative transmembrane domains. By gene disruption we demonstrate that the sts1+ gene is not essential for viability. These disruptants exhibit pleiotropic defects, such as cold-sensitivity for growth and at the permissive temperature, a supersensitivity to divalent cations and several unrelated drugs including staurosporine, caffeine, chloramphenicol, sorbitol, and SDS. Disruption of the sts1+ gene does not lead to a sensitivity to thiabendazole or hydroxyurea.     

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.     

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2⁺ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2⁺; overexpression of atb2⁺ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.     

C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast <Emphasis Type="Italic">S</Emphasis>c<Emphasis Type="Italic">hizosaccharomyces pombe</Emphasis>

The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-?CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.     

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.