Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast.

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30 degrees C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37 degrees C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast.

The Schizosaccharomyces pombe dhp1(+) gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5'-->3' exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)(+) RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1(+) is required for proper chromosome segregation as well as for poly(A)(+) RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1(+). We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism.

We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.

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.

Intramedullary spinal cord metastasis from gastric cancer. Case report.

A case of intramedullary spinal cord metastasis from gastric cancer is reported. A 51-year-old woman presented with hemicord syndrome that had progressed within 1 month to tetraplegia. Despite total resection of the tumor, she died of disseminated intravascular coagulation and multiple organ failure. Examination of pathological findings demonstrated undifferentiated adenocarcinoma, and postoperative gastroendoscopic study revealed advanced gastric cancer. To the authors' knowledge this is the first case of intramedullary spinal cord metastasis from gastric cancer. The clinical characteristics of the disease are discussed.

[Acute cardiac tamponade in nonpenetrating injury of proximal aorta: report of a case].

A 21-year-old male admitted to the emergency room with multiple traumatic injuries and shock. Although a chest X-ray showed normal in size of cardiac shadow, echocardiography confirmed cardiac tamponade. A median sternotomy was performed and the pericardial space was evacuated of 600 ml of blood and blood clots. Minor bleeding was identified in the proximal aorta, and complete hemostasis was achieved with manual compression and fibrin glue sealing. One month follow-up showed no pseudoaneurysm formation.

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe.

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1+ gene was present in the introduced leu1::ura4+ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of non-homologous to homologous recombination events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites. Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.

The budding yeast cohesin gene SCC1/MCD1/RHC21 genetically interacts with PKA, CDK and APC.

Cohesin is a protein that plays a key role in the cohesion and separation of sister chromatids. During the duplication of chromatids, cohesin holds sister chromatids together until the onset of anaphase, and thereby prevents the premature separation of sister chromatids which would otherwise jeopardize the faithful segregation of chromosomes. To investigate the molecular mechanisms of sister chromatid cohesion, we have isolated multicopy suppressors of a temperature-sensitive (ts) mutation in the SCC1/MCD1/RHC21 gene which encodes a component of the cohesin complex in budding yeast. Isolation of multicopy suppressors of rhc21-sk16 and further genetic analyses revealed that several distinct biological pathways are involved in the regulation of SCC1/MCD1/RHC21 function. Firstly, PDE2 and BCY1, each of which inhibits the activity of protein kinase A (PKA), suppressed the temperature sensitivity of the rhc21-sk16 mutant. Secondly, PDE2 suppressed the temperature sensitivity of the cdc16-1 mutant. These results suggest that SCC1/MCD1/RHC21 is negatively regulated by the PKA pathway via the anaphase promoting complex (APC). Thirdly, ZDS1, a multicopy suppressor of cdc28-1N, and its homologue ZDS2 were isolated as multicopy suppressors of rhc21-sk16. Furthermore, the rhc21-sk16 mutant did not grow in the presence of the cdc28-1N mutation. Hence, SCC1/MCD1/RHC21 is positively regulated by the mitotic CDK, CDC28. Finally, SCC1/MCD1/RHC21 was found to interact genetically with CDC20, an activator of APC. Overexpression of CDC20 suppressed the temperature sensitivity of rhc21-sk16, and rhc21-sk16 was shown to be synthetically lethal with cdc20-1. In addition, the growth of the rhc21-sk16 mutant was inhibited by overproduction of the anaphase inhibitor Pds1p, whose degradation is mediated by Cdc20p in APC-dependent proteolysis. The functional relationships between SCC1/MCD1/RHC21 and PKA, CDK or APC are discussed.

The RHC21 gene of budding yeast, a homologue of the fission yeast rad21+ gene, is essential for chromosome segregation.

The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21+ gene, which affects the sensitivity of cells to gamma-irradiation and is essential for cell growth in S. pombe. Disruption of the RHC21 gene showed that it is also essential in S. cerevisiae. To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16. When this mutant was incubated at 36 degrees C, the percentage of large-budded cells was increased. Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell. Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25 degrees C. Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant. These results indicated that the RHC21 gene is required for proper segregation of the chromosomes. In addition, we found that the mutant is sensitive not only to UV radiation and gamma-rays but also to the antimicrotubule agent nocodazole at 25 degrees C. This suggests that the RHC21 gene is involved in the microtubule function. We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function.

Bloom's syndrome gene suppresses premature ageing caused by Sgs1 deficiency in yeast.

BACKGROUND: Bloom's syndrome (BS) is an autosomal recessive disorder causing short stature, immunodeficiency, and an increased risk of cancer. Increased rates of sister chromatid exchange and chromosomal aberration have been observed in cells having defects in the BLM gene. Among five kinds of human RecQ helicases cloned, the mutations in WRN and RecQL4 have been known as the causes of premature ageing. Little is, however, known about the function of BLM helicase in ageing. RESULTS: We show that human BLM, but not WRN can prevent the premature ageing and the increased homologous recombination at the rDNA loci caused by sgs1 mutation. Unexpectedly, the levels of ERCs (extrachromosomal rDNA circles), the products of homologous recombination, formed in 7-generation cells of the wild-type or the sgs1:BLM strain were comparable with those of the sgs1 or the sgs1:WRN age-matched-old cells. CONCLUSION: These results imply that BLM helicase may have an important role in human ageing. In addition, these data suggest that the accumulated ERCs per se may be not the cause of premature ageing in yeast, inconsistent with the model proposed by Sinclair & Guarente. We discuss a new model, which explains how Sgs1 or BLM helicase suppresses premature ageing in yeast.