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.

Fission yeast centromeres vary in size but are organized in a similar fashion. Each consists of two distinct domains, namely, the -15-kilobase (kb) central region (cnt + imr), containing chromosome-specific low copy number sequences, and 20-to 100-kb outer surrounding sequences (otr) with highly repetitive motifs common to all centromeres. The central region consists of an inner asymmetric sequence flanked by inverted repeats that exhibit strict identity with each other. Nucleotide changes in the left repeat are always accompanied with the same changes in the right. The chromatin structure of the central region is unusual. A nucleosomal nuclease digestion pattern formed on unstable plasmids but not on stable chromosome. DNase I hypersensitive sites correlate with the location of tRNA genes in the central region. Autonomously replicating sequences are also present in the central region. The behavior of truncated minichromosomes suggested that the central region is essential, but not sufficient, to confer transmission stability. A portion of the outer repetitive region is also required. A larger outer region is necessary to ensure correct meiotic behavior. Fluorescence in situ hybridization identified individual cens. In the interphase, they cluster near the nuclear periphery. The central sequence (cnt + imr) may play a role in positioning individual chromosomes within the nucleus, whereas the outer regions (otr) may interact with each other to form the higher-order complex structure.

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.

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3 ؉ gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3⌬ cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found that pmt3 ؉ is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase-and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.Ubiquitin is a small (76-residue), abundant protein conserved in all eukaryotic cells. It exists in several cellular compartments, such as the cytosol, nucleus, and cell surface. It is well known that ubiquitin regulates the function and stability of target proteins through its posttranslational conjugation to target proteins. Before conjugation to target proteins, ubiquitin must be processed by a C-terminal hydrolase. The first step of the ubiquitin conjugation pathway is the ATP-dependent formation of a thioester bond between the conserved C-terminal glycine of processed ubiquitin and the active-site cysteine residue of an E1 ubiquitin-activating enzyme. The second step is the transfer of activated ubiquitin to the active-site cysteine of an E2 ubiquitin-conjugating enzyme. In the final step, the E2 enzyme may cooperate with an E3 ubiquitin protein ligase to form an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino groups of lysine residues of target proteins. Ubiquitin covalently conjugated to target proteins can be removed by a ubiquitin isopeptidase (89).Recently, a number of novel ubiquitin-like proteins were independently discovered in a number of species, suggesting that ubiquitin is part of a family of related proteins involved in the covalent modification of proteins. The first example of such a protein was the 15-kDa interferon-inducible, ubiquitin crossreacting protein UCRP (25). UCRP contains two ubiquitinr...

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