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Aneuploidy decreases cellular fitness, yet it is also associated with cancer, a disease of enhanced proliferative capacity. To investigate one mechanism by which aneuploidy could contribute to tumorigenesis, we examined the effects of aneuploidy on genomic stability. We analyzed 13 budding yeast strains that carry extra copies of single chromosomes and found that all aneuploid strains exhibited one or more forms of genomic instability. Most strains displayed increased chromosome loss and mitotic recombination, as well as defective DNA damage repair. Aneuploid fission yeast strains also exhibited defects in mitotic recombination. Aneuploidy-induced genomic instability could facilitate the development of genetic alterations that drive malignant growth in cancer.

Recent advances in metabolomics technology have enabled large-scale comprehensive analyses of metabolites, but the throughput of data processing of non-targeted, quantitative differential analyses is very low. It is crucial to solve this problem to generate biological hypotheses from a large-scale dataset. To improve the analysis of metabolite data, we focused on the processing of quantitative differential analysis after multiple peak alignment. We have developed a program named FAQuant that automatically selects reliable peaks from each chromatogram, quantifies the mean of peak intensity to compare between sample groups, and selects the peaks with differences in accumulation of metabolites. This program was incorporated into a quantitative differential metabolome pipeline as a module to improve the throughput of gas chromatography-mass spectrometry dataset analysis. As a result, the module incorporation largely reduced the total processing time. Furthermore, differential analysis of metabolites in soybean (Glycine max) cultivars was demonstrated by use of the system. This system might facilitate biological hypothesis generation from large-scale comparative metabolome analysis.

A previously isolated fission yeast γ-tubulin mutant containing apparently stabilized microtubules proliferated at an approximately identical rate as wild type, yet the mutant mitosis spindle dynamics were aberrant, particularly the kinetochore microtubule dynamics. Progression through mitosis in the mutant, however, resulted in mostly accurate chromosome segregation. In the absence of the spindle assembly checkpoint gene, mad2+, the spindle dynamics in the γ-tubulin mutant were greatly compromised, leading to a high incidence of chromosome missegregation. Unlike in wild-type cells, green fluorescent protein (GFP)-tagged Mad2 protein often accumulated near one of the poles of an elongating spindle in the γ-tubulin mutant. We isolated novel mad2 mutants that were defective in arresting mitotic progression upon gross perturbation of the spindle formation but remained functional for the viability of the γ-tubulin mutant. Further, the mad2 mutations did not appreciably destabilize minichromosomes in unperturbed mitoses. When overexpressed ectopically, these mutant Mad2 proteins sequestered wild-type Mad2, preventing its function in mitotic checkpoint arrest, but not in minichromosome stability. These results indicated that the Mad2 functions required for checkpoint arrest and chromosome stability in unperturbed mitosis are genetically discernible. Immunoprecipitation studies demonstrated that GFP-fused mutant Mad2 proteins formed a Mad1-containing complex with altered stability compared to that formed with wild-type Mad2, providing clues to the novel mad2 mutant phenotype.

In interphase cells of fission yeast, the spindle pole body (SPB) is thought to be connected with chromosomal centromeres by an as yet unknown mechanism that spans the nuclear membrane. To elucidate this mechanism, we performed two-hybrid screens for proteins that interact with Kms1 and Sad1, which are constitutive membrane-bound components of the SPB that interact with each other. Seven and 26 genes were identified whose products potentially interact with Kms1 and Sad1, respectively. With the exception of Dlc1 (a homolog of the 14-kDa dynein light chain), all of the Kms1 interactors also interacted with Sad1. Among the genes identified were the previously known genes rhp9 +/ crb2 +, cut6 +, ags1 +/ mok1 +, gst3 +, kms2 +, and sid4 +. The products of kms2 + and sid4 + localize to the SPB. The novel genes were characterized by constructing disruption mutations and by localization of the gene products. Two of them, putative homologues of budding yeast UFE1 (which encodes a t-SNARE) and SFH1 (an essential component of a chromatin-remodeling complex), were essential for viability. Two further genes, which were only conditionally essential, genetically interact with sad1 + . One of these was named sif1 + (for Sad1-interacting factor) and is required for proper septum formation at high temperature. Cells in which this gene was overexpressed displayed a wee -like phenotype. The product of the other gene, apm1 +, is very similar to the medium chain of an adaptor protein complex in clathrin-coated vesicles. Apm1 appears to be required for SPB separation and spindle formation, and tended to accumulate at the SPB when it was overproduced. It was functionally distinct from its homologues Apm2 and Apm4. Other novel genes identified in this study included one for a nucleoporin and genes encoding novel membrane-bound proteins that were genetically related to Sad1. We found that none of the newly identified genes tested were necessary for centromere/telomere clustering.

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1+ gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1+ gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.

We have isolated a mutant of Schizosaccharomyces pombe whose growth is temperature sensitive when it is haploid but not when it is diploid. This mutant may provide a useful system for selecting nonconditional mutants which are defective in diploid formation upon conjugation.

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability., Author Summary Aneuploidy is a major cause of abortive development and is implicated in tumorigenesis in humans. Recent studies revealed that the increased need for protein degradation might account for the detrimental effects of aneuploidy on a cell. Here, we investigated the genetic systems responsible for aneuploid viability. Using a collection of gene deletions in fission yeast, we isolated mutants that affect aneuploid viability. We found that an evolutionarily conserved transcription regulator, the CCR4-NOT complex, and its related factors are required for aneuploid viability, suggesting that regulation of mRNA turnover is required to tolerate aneuploidy. In addition, homologous recombination repair is important for aneuploid viability.

The core proteins of the spindle assembly checkpoint (SAC), Mads, Bubs, and Mps1, first identified in the budding yeast, are thought to be functionally and structurally conserved through evolution. We found that fission yeast Bub3 is dispensable for SAC, as bub3 null mutants blocked mitotic progression when spindle formation was disrupted. Consistently, the bub3 mutation only weakly affected the stability of minichromosome Ch16 compared with other SAC mutants. Fission yeast Rae1 has sequence homology with Bub3. The bub3 rae1 double mutant and rae1 single mutant did not have defective SAC, suggesting that these genes do not have overlapping roles for SAC. Observations of living cells revealed that the duration of the mitotic prometaphase/metaphase was longer in the bub3 mutant and was Mad2 dependent. Further, the bub3 mutant was defective in sister centromere association during metaphase. Together, these findings suggest that fission yeast Bub3 is required for normal spindle dynamics, but not for SAC.

Aneuploid generation and stability are biologically important. In the present study, we investigated fission yeast aneuploids, focusing on the process through which aneuploidy is resolved into stable euploidy. The viability and growth patterns of aneuploid spores were greatly influenced by culture conditions, including nutrition and temperature. Germ tube formation and DNA synthesis in a major portion of aneuploids were greatly delayed or arrested. Observation of individual spores and their growth profiles revealed that a certain type(s) of aneuploid resolved its aneuploidy into normal euploids through anomalous cell divisions, which in many cases produced dead cells. Another type of aneuploid, disomy of chromosome 3, the only maintainable aneuploid between n and 2n, showed a peculiar cell division arrest phenotype under a certain growth condition. Microcolonies that formed from this type of aneuploid often contained a population of cells that became incompetent for cell division. This cell division arrest was not due to a nutritional limitation. During this peculiar process of colony formation, stable haploids or diploids were frequently produced. All other types of aneuploids are usually inviable, at least under our experimental conditions. To examine the aneuploid issue more systematically, we constructed a system to select for disomy of chromosome 1 or 2 using intragenic complementation of ade6-M210 and -M216 alleles. This genetic selection system revealed that fission yeast aneuploids can be stabilized through structural chromosome changes, including partial duplication and circular mini-chromosomes.

Cloning and sequencing of the ure1 + gene of Schizosaccharomyces pombe indicated that it encodes the urease which had been biochemically identified. The fission yeast urease has a one-subunit structure like those from plants but different from bacterial ureases which are composed of two or three distinct subunits. Genetic analyses showed that the ure1 + gene product is actually involved in urea metabolism.

In fission yeast, γ-tubulin (encoded by the gtb1+ gene), Alp4 (Spc97/GCP2), and Alp6 (Spc98/GCP3) are essential components of the γ-tubulin complex. We isolated gtb1 mutants as allele-specific suppressors of temperature-sensitive alp4 mutations. Mutation sites in gtb1 mutants and in several alp4 alleles were determined. The majority of substituted amino acids were mapped to a small area on the predicted surface of the γ-tubulin molecule that might directly interact with the Alp4 protein. The cold sensitivity of γ-tubulin mutants was almost completely suppressed by an α-tubulin mutation and partially suppressed by a low concentration of thiabendazole, a microtubule assembly inhibitor. Other gtb1 mutants had increased resistance to this drug. Gel-filtration and immunoprecipitation analyses suggested that the mutant γ-tubulin formed an altered γ-tubulin complex with increased stability compared to wild-type γ-tubulin. In most gtb1 mutants, sexual development was impaired, and aberrant asci that contained an irregular spore shape and number were produced. In contrast, spore formation was not appreciably damaged in some alp4 and alp6 mutants, even at temperatures where vegetative proliferation was substantially defective. These results suggested that the function of the γ-tubulin complex or the requirement of each component of the complex is differentially regulated between the vegetative and sexual phases of the life cycle in fission yeast. In addition, genetic data indicated intimate functional connections of γ-tubulin with several kinesin-like proteins.