Search

Bortezomib/PS-341/Velcade, a proteasome inhibitor, is widely used to treat multiple myeloma. While several mechanisms of the cytotoxicity of the drug were proposed, the actual mechanism remains elusive. We aimed to identify genes affecting the cytotoxicity of Bortezomib in the fission yeast S. pombe as the drug inhibits this organism's cell division cycle like proteasome mutants. Among the 2815 genes screened (covering 56% of total ORFs), 19 genes, whose deletions induce strong synthetic lethality with Bortezomib, were identified. The products of the 19 genes included four ubiquitin enzymes and one nuclear proteasome factor, and 13 of them are conserved in humans. Our results will provide useful information for understanding the actions of Bortezomib within cells.

The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that approximately 850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype.

Fission yeast Schizosaccharomyces pombe temperature-sensitive (ts) cut1 mutants fail to separate sister chromatids in anaphase but the cells continue to divide, leading to bisection of the undivided nucleus (the cut phenotype). If cytokinesis is blocked, replication continues, forming a giant nucleus with polyploid chromosomes. We show here that the phenotype of ts cut2-364 is highly similar to that of cut1 and that the functions of the gene products of cut1+ and cut2+ are closely interrelated. The cut1+ and cut2+ genes are essential for viability and interact genetically. Cut1 protein concentrates along the short spindle in metaphase as does Cut2. Cut1 (approximately 200 kDa) and Cut2 (42 kDa) associate, as shown by immunoprecipitation, and co-sediment as large complexes (30 and 40S) in sucrose gradient centrifugation. Their behavior in the cell cycle is strikingly different, however: Cut2 is degraded in anaphase by the same proteolytic machinery used for the destruction of cyclin B, whereas Cut1 exists throughout the cell cycle. The essential function of the Cut1-Cut2 complex which ensures sister chromatid separation may be regulated by Cut2 proteolysis. The C-terminal region of Cut1 is evolutionarily conserved and similar to that of budding yeast Esp1, filamentous fungi BimB and a human protein.

Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division. Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed. Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth. The sts5 mutant is not defective in cell wall integrity. Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal. Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation. Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation. The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis. The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants. Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed. Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting. A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure. These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.

Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.

Fission yeast pap1+ gene encodes an AP-1-like transcription factor, whose overexpression can confer resistance to staurosporine, a protein kinase inhibitor. We have previously identified a target gene (p25) for pap1+, and shown that, crm1+, which is required for maintenance of higher order chromosome structure, negatively regulates pap1-dependent transcription. In this study, we have characterized a novel gene, pad1+, which was isolated as a multicopy plasmid capable of conferring staurosporine-resistance. We showed that high copy pad1+ induces transcriptional activation of the p25 gene and that the induction by pad1+ is dependent on the pap1+ gene. Furthermore, a cis-element analysis of the 5'-region of the p25 gene showed that two elements (an AP-1 site and a 14 bp palindrome sequence) where pap1 binds in vitro is essential for the induction by pad1+. These results indicate that pad1 can positively regulate pap1-dependent transcription. Through an electromobility shift assay we showed that overexpression of pad1+ is not capable of enhancing the DNA-binding activity of pap1 directly. The pad1+ gene encodes a 35 kDa protein that has significant identity (68%) to Caenorhabditis elegans F37A4.5, and is also similar to mouse Mov34 and human C6.1A. Gene disruption experiments have demonstrated that pad1+ is essential for viability. A disruption mutant of pad1+ obtained after spore germination exhibited an elongated cell body with abberantly folded chromosomes. A mitotic plasmid loss experiment also produced similar cells having an abnormal chromosome structure. These suggest that pad1+ may play an important role in higher order chromosome structure. Taken concurrently with our previous results, two essential genes pad1+ and crm1+ regulate pap1-dependent transcription; pad1+ and crm1+ are positive and negative regulators, respectively.

A novel anaphase block phenotype was found in fission yeast temperature-sensitive cut9 mutants. Cells enter mitosis with chromosome condensation and short spindle formation, then block anaphase, but continue to progress into postanaphase events such as degradation of the spindle, reformation of the postanaphase cytoplasmic microtubule arrays, septation, and cytokinesis. The cut9 mutants are defective in the onset of anaphase and possibly in the restraint of postanaphase events until the completion of anaphase. The cut9+ gene encodes a 78-kD protein containing the 10 34-amino acid repeats, tetratricopeptide repeats (TPR), and similar to budding yeast Cdc16. It is essential for viability, and the mutation sites reside in the TPR. The three genes, namely, nuc2+, scn1+, and scn2+, genetically interact with cut9+. The nuc2+ and cut9+ genes share an essential function to initiate anaphase. The cold-sensitive scn1 and scn2 mutations, defective in late anaphase, can suppress the ts phenotype of cut9.

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.

A calcineurin (type 2B)-like protein phosphatase gene designated ppb1+ was isolated from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence was 57% identical to rat PP2B alpha. ppb1 null mutant could form colonies at 33 degrees C but the size of the colonies was small at 22 degrees C. Cytokinesis was greatly delayed at 22 degrees C, and a large number of multi-septate cells were produced. The cell polarity control was impaired, causing branched cells. ppb1 null was virtually sterile. These phenotypes were rescued by a plasmid carrying the ppb1+ gene. Multi-septate cells were also produced in wild type at 22 degrees C by cyclosporin A, an inhibitor of calcineurin. This drug effect was enhanced in stst1 null mutant, which was hypersensitive to various drugs and cations. ppb1 null was not affected by cyclosporin A, consistent with the hypothesis that ppb1 is its target. Double-mutant analysis indicated that ppb1 had a function related to that of two other phosphatases, type 1-like dis2 and 2A-like ppa2.ppb1 null-sts1 null showed the severe multi-septate phenotype in the absence of cyclosporin A. ppb1+ and sts1+ gene functions are related. The double mutant ppb1-sts5 was lethal, indicating that the ppb1+ gene shared an essential function with the sts5+ gene. Overexpression of ppb1+ caused anomalies in cell and nuclear shape, microtubule arrays and spindle pole body positioning in interphase cells. Thus the ppb1+ gene appears to be involved in cytokinesis, mating, transport, nuclear and spindle pole body positioning, and cell shape.

Two novel protein kinase C (n PKC) gene homologues, pck1+ and pck2+ were isolated from the fission yeast Schizosaccharomyces pombe (Toda et al. (1993) EMBO J. 12, 1987). We examined the functional differences of pck1+ and pck2+ in cell wall formation and actin organization of S. pombe. Regenerating protoplasts of a wild-type strain, single gene disruptants of pck1+ (delta pck1) and pck2+ (delta pck2) were used as a simple model to examine the functional links between PKC, cell wall formation and actin organization. Protoplasts of the wild-type strain and those of delta pck1 reverted to intact cells in osmotically stabilized liquid medium. A close spatial association between new cell wall formation and actin was observed in these two strains. In delta pck2, protoplasts did not revert to intact cells: (1) scarcely any new cell wall material was formed; (2) actin was not reorganized; and (3) nuclear division and an increase in the amount of cytoplasm were observed in the regenerating protoplasts. These findings demonstrate that the pck2+ gene has a function essential for protoplast regeneration but the pck1+ gene does not. Involvement of n PKCs in cell wall formation and actin organization was also clarified. The effect of staurosporine (a potent inhibitor of protein kinases) on regenerating protoplasts of the three strains confirmed the assumption that the pck2 protein is an in vivo target of staurosporine in the fission yeast.

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.

Fission yeast cut5 mutants cause cytokinesis in the absence of normal nuclear division. We show here that cut5+ is required for both the onset of S phase and the restraint of M phase before the completion of S phase. The primary defects in cut5 mutants occur prior to S phase, but cells suffer lethal damage during M phase. Mitosis and cytokinesis occur in the presence of hydroxyurea or in the double mutant cdc10-cut5 (the cdc10 mutation alone blocks progression from G1 to S). Gene cloning shows that cut5+ is identical to the fission yeast rad4+ gene, which is similar to human XRCC1. The rad4+/cut5+ gene is unique in its positive role for replication/repair and in its negative role for mitosis/cytokinesis. We propose a single/twin chromatid marking model for rad4+/cut5+ function in cell cycle control.

Two novel protein kinase C (PKC)-like genes, pck1+ and pck2+ were isolated from fission yeast by PCR. Both contain common domains of PKC-related molecules, but lack a putative Ca(2+)-binding domain so that they may belong to the nPKC group. Gene disruption of pck1+ and pck2+ establishes that they share an overlapping essential function for cell viability. Cells of a single pck2 deletion display severe defects in cell shape; they are irregular and sometimes pear-like instead of cylindrical. In contrast, the induced overexpression of pck2+ is lethal, producing multiseptated and branched cells. These results suggest that fission yeast PKC-like genes are involved in the polarity of cell growth control. We show that pck2 is allelic to sts6, a locus we have previously identified by its supersensitivity to staurosporine, a potent protein kinase inhibitor [Toda et al. (1991) Genes Dev., 5, 60-73]. In addition, the lethal overexpression of pck2+ can be suppressed by staurosporine, indicating that fission yeast pck1 and pck2 are molecular targets of this inhibitor.

We isolated a fission yeast putative protein serine/threonine phosphatase gene designated ppe1+ by hybridization. The predicted amino acid sequence is similar to those of the fission yeast ppa2 (53% identity) and dis2 (39%) phosphatases, and highly similar to those of the budding yeast SIT4 (72%), Drosophila PPV (68%) and rabbit PPX (61%) phosphatases. Antibodies against ppe1 protein identified a 37-kd polypeptide in fission yeast. A gene disruption (designated delta ppe1) caused cold-sensitive lethality and short, pear-shaped cells. These phenotypes were fully suppressed by a plasmid carrying ppe1+. Three classes of multicopy suppressor genes for delta ppe1 were identified as follows: 1) ppa1+ and ppa2+ encoding type 2A-like phosphatases, 2) mitotically essential dis3+ similar to the budding yeast SSD1/SRK1, a suppressor for sit4, and 3) pck1+ coding for a protein kinase C-like kinase. Consistently, the budding yeast SIT4 gene was also a multicopy suppressor for delta ppe1. Phosphatase ppe1 may play a role in cell morphogenesis and mitosis by either regulating or being regulated by these multicopy suppressor gene products. Consistent with this hypothesis, double mutants ppe1-ppa2 and ppe1-pck1 are lethal at the permissive temperature.

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.

The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis.

The recently discovered TPR gene family encodes a diverse group of proteins that function in mitosis, transcription, splicing, protein import and neurogenesis. These multi-domain proteins all contain tandemly arranged repeats of a 34-amino acid motif that are presumed to form helix-turn structures, each with a 'knob' and 'hole', acting as helix-associating domains.

We report here that the fission yeast centromere regions in the three chromosomes contain no less than 36 symmetrically arranged tRNA-coding sequences, and many of them are located within the inner inverted regions that are thought to be essential for the centromere function. There are 11 different species of tRNA-coding sequences, and four of them are identical to those previously known in this organism. This high-density distribution of tRNA genes in the centromere regions is surprising, as the fission yeast centromeres were thought to form transcriptionally inactive structures.

The fission yeast dis2+ gene encodes one of the two type 1 protein phosphatases (PP1) in this organism. Its semidominant mutant dis2-11 is defective in mitosis. Here we report the characterization of a high dosage suppressor, sds22+, that complements dis2-11. Sequencing of the cloned sds22+ gene predicts a novel 30 kd protein, which consists almost entirely of leucine-rich 22 amino acid repeats and is enriched in the insoluble nuclear fraction. sds22+ is an essential gene required for the mitotic metaphase/anaphase transition; gene disruption causes cell cycle arrest at midmitosis. Unexpectedly, the sds22+ gene becomes dispensable upon high dosage of the PP1 genes. The sds22+ product appears to facilitate PP1-dependent dephosphorylation, but does not substitute PP1. We propose that the sds22+ protein forms a repeating helical rod that is capable of enhancing a PP1-dependent dephosphorylation activity that is essential in midmitosis.

The activities of type 1 protein phosphatase (PP1) and 2A (PP2A) have distinct, essential roles in cell cycle control. Two previously identified PP1 genes (dis2+ and sds21+) and two PP2A genes (ppa1+ and ppa2+), highly homologous to mammalian PP2A, have been isolated from fission yeast. Only double gene disruption of both PP2A genes results in lethality, as is the case for PP1 genes. By fractionating and assaying PPases in wild-type, various deletion, and point mutant strains, the decrease of PP1 or PP2A activity is shown to cause mitotic defects, exhibiting strikingly different cell cycle phenotypes: cold-sensitive mutations in the same amino acid lesion of PP1 and PP2A produce chromosome nondisjunction and premature mitosis, respectively. Consistently, PP1 and PP2A genes cannot be functionally substituted. Although the overall levels of PP1 and PP2A activities do not fluctuate during the cell cycle, subpopulations might be regulated.

The structure equivalent to higher eukaryotic centrosomes in fission yeast, the nuclear membrane-bound spindle pole body, is inactive during interphase. On transition from G2 to M phase of the cell cycle, the spindle pole body duplicates; the daughter pole bodies seed microtubules which interdigitate to form a short spindle that elongates to span the nucleus at metaphase. We have identified two loci which, when mutated, block spindle formation. The predicted product of one of these genes, cut7+, contains an amino-terminal domain similar to the kinesin heavy chain head domain, indicating that the cut7+ product could be a spindle motor. The cut7+ gene resembles the Aspergillus nidulans putative spindle motor gene bimC, both in terms of its organization with a homologous amino-terminal head and no obvious heptad repeats and in the morphology of the mutant phenotype. But we find no similarity between the carboxy termini of these genes, suggested that either the cut7+ gene represents a new class of kinesin genes and that fission yeast may in addition contain a bimC homologue, or that the carboxy termini of these mitotic kinesins are not evolutionarily conserved and that the cut7+ gene belongs to a subgroup of bimC-related kinesins.

Mutations in the fission yeast cut1+, cut2+, and cut10+ genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis. DNA synthesis continues, making polyploid nuclei with several spindles. Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells. The cut1+ and cut2+ genes are cloned by transformation. High gene dosage of cut1+ also complements cut2 and cut10 mutants. The cut2+ gene, however, complements only cut2. The 210 kd cut1+ gene product contains putative ATP binding and helical coil regions followed by a COOH-terminal domain homologous to the S. cerevisiae gene ESP1. Mutations in the ESP1 gene also result in many SPBs. The cut1+ product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction. It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.

The S. pombe nuc2+ gene is required for mitotic chromosome disjunction. Its mutation arrests mitosis at the metaphase. The gene product is present in the nuclear scaffold-like fraction. The nuc2+ protein contains a domain, separated from ten 34 amino acid repeat segments, that is capable of binding AT-rich DNA in vitro. The ts mutation resides in one of the 34 amino acid repeats. Circular dichroism, limited proteolysis of the repeats, and model fitting indicate the presence of helical segments connected by protease-sensitive hinges. We propose that these repeats form a novel secondary structure (snap helix) having "knob and hole" helix-associating motifs. The packing of the snap helices would be stabilized by the bonding between the hydrophobic amino acids surrounding the knobs and holes. The nuc2+ protein may in one way bind to DNA and in another way mutually associate to form a part of the chromosome scaffold.

S. pombe centromeres are large and complex. We introduced a method that enables us to characterize directly centromere DNAs. Genomic DNA fragments containing cen1, cen2, or cen3, respectively, are made by cleaving NotI sites integrated on target sites and are partially restricted for long-range mapping in PFG electrophoresis. The 40 kb long cen1 consists of two inverted approximately 10 kb motifs, each containing centromeric elements dg and dh, flanked by a central region. In cen2, three motifs are arranged in inverted and direct orientations with flanking domains, making up the approximately 70 kb long repetitious region. In cen3, approximately 15 copies of dg-dh constitute a region longer than 100 kb. A set of inverted motifs with an approximately 15 kb central region might be a prototype for the S. pombe centromeres. The motifs appear to play a role in chromosome stability and segregation. Their action may be additive, and the mutual directions of dg and dh inside a motif may not be essential for function.

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.

We isolated a novel class of Schizosaccharomyces pombe cold-sensitive mutants with deformed nuclear chromosome domains consisting of thread- or rodlike condensed segments at restrictive temperature. Their mutations were mapped in a novel, identical locus designated crm1 (chromosomal region maintenance). The crm1 mutants also show the following phenotypes. DNA, RNA, and protein syntheses diminish at restrictive temperature. At permissive temperature, the amount of one particular protein, p25, greatly increases. The mutant growth is hypersensitive to Ca2+ and resistant to protein kinase inhibitors. We cloned the 4.1-kb-long crm1+ gene that rescued the above phenotypes by transformation and determined its nucleotide sequence, which predicts a 1,077-residue protein. Affinity-purified antiserum raised against the crm1+ polypeptide expressed in Escherichia coli detected a 115-kD protein in S. pombe extracts. Genomic Southern hybridization and immunoblotting suggested that the crm1+ product might be highly conserved in distant organisms. Through immunofluorescence microscopy, the crm1+ protein appeared to be principally localized within the nucleus and also at its periphery. We speculate that the crm1+ protein might be one of those nuclear components that modify the chromosome structures or regulate the nuclear environment required for maintaining higher order chromosome structures.

The cells of a cold-sensitive mutant nda3-KM311 of the fission yeast Schizosaccharomyces pombe were arrested highly synchronously at a step similar to mitotic prophase when incubated at a restrictive temperature. DAPI staining and indirect immunofluorescence microscopy showed three condensed chromosomes but no spindle. Six minutes after the temperature shifted to a permissive one, the spindle appeared and elongated. The chromosomes were separated at a constant speed (relative velocity 1 micron/min), and the spindle disappeared after the chromosomes reached opposite ends of the cell. The NDA3 gene of S. pombe was cloned by transformation. The 2.6 kb Hind III genomic DNA that complemented the nda3 mutations had only one coding frame split with five short introns. The predicted amino acid sequence contained 448 residues, and was 75% homologous to that of chicken beta-tubulin.

S. pombe dis mutants block mitotic chromosome disjunction in a manner reminiscent of aneuploidy formation, and belong to three distinct genes, dis1-dis3. We cloned two independent genomic DNAs that complemented both the cold-sensitive and caffeine-hypersensitive phenotype of dis2-11. These genes, dis2+ and a suppressor sds21+, encode proteins (calculated MW 37,000) with similar predicted amino acid sequences. dis2+ and sds21+ have overlapping functions, and disruptants are lethal only when both genes are disrupted. The gene products identified by anti-dis2 serum are enriched in nuclei. By hybridization, we obtained two cDNA clones from mouse and one genomic clone from S. cerevisiae; the latter complements S. pombe dis2-11. These dis2+ and similar polypeptides of yeasts and mouse are found to be highly homologous (75%-90% identical) to rabbit protein phosphatase 1. The implications of these findings are discussed with regard to mitotic control.

Mutations in a cell-cycle gene NDA2 of Schizosaccharomyces pombe have pleiotropic effects on nuclear division, nuclear location, and thiabendazole sensitivity ( Toda et al., 1983). By transformation and nucleotide sequence determination, we identified NDA2 as one of two alpha-tubulin genes present in the genome of S. pombe. Two cloned sequences complemented cold-sensitive and thiabendazole-supersensitive nda2 mutations; one was derived from NDA2 that encodes alpha 1-tubulin, the other from an unidentified locus encoding alpha 2-tubulin. The predicted amino acid sequences showed that the alpha 1- and alpha 2-tubulins had respective residues of 455 and 449 (molecular weights 51,200 and 50,600). The homology to porcine alpha-tubulin was 76% in both cases. Frequent alterations took place in the two restricted regions. The alpha 1-tubulin (NDA2) clone had a 90 bp intervening sequence, the alpha 2-tubulin clone did not. RNA blot hybridization experiments indicated that both genes are transcribed. S. pombe tubulin was isolated by cycles of assembly and disassembly. Presumed alpha- and beta-tubulin polypeptide bands reacted with monoclonal antibodies specific for chicken alpha- and beta-tubulins.

Gene products required for mitotic chromosome separation in the fission yeast Schizosaccharomyces pombe are described. They have been identified by two distinct strategies of mutant isolation, followed by gene cloning and immunochemical characterization of gene products. The roles of four representative genes, namely nda3+, nuc2+, top2+ and dis2+, encoding beta-tubulin, a nuclear scaffold-like protein, DNA topoisomerase II and type-1 protein phosphatase, respectively, are discussed in regard to the mechanisms and control of chromosome separation.

A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization. We cloned the nuc1+ gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I. We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (approximately 190 kD and 2 x 10(4) copies/cell) in the S. pombe nuclear fraction. By immunofluorescence microscopy, anti-nuc1+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus. The nucleolar immunofluorescence by anti-nuc1+ was faded in nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II. Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.

We cloned the structural gene topl+ for Schizosaccharomyces pombe DNA topoisomerase I (topo I) by hybridization. An eight-fold increase of topo I relaxing activity was obtained in S. pombe cells transformed with multicopy plasmid with topl+ insert. Nucleotide sequence determination showed a hypothetical coding frame interrupted by two short introns, encoding a 812 residue polypeptide (M.W. 94,000), 43 residues longer than and 47% homologous to Saccharomyces cerevisiae topo I. We show that the topl (null) strain made by gene disruption is viable, although its generation time is 20% longer than that of wild type. The topl locus is mapped in the long arm of chromosome II, using the Leu+ marker integrated with the cloned topl+ sequence. We constructed a double mutant topl (null) top2 (ts) and found its defective phenotype similar to that of previously obtained topl (heat sensitive) top2 (ts). The other double mutant topl (null) top2 (cs), however, was lethal. Our results suggest that topl+ gene of S. pombe is dispensable only if topo II activity is abundant.

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe. There are 14 detectable Not I sites in S. pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III. The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis. These fragments ranged in size from 4.5 kb to approximately 3.5 Mb. Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes. The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb. These are generally somewhat smaller than estimated previously.