Keita Aoki

Publications (8)56.63 Total impact

• Article: Construction of an insertion marker collection of Sz. japonicus (IMACS) for genetic mapping and a fosmid library covering its genome.

  Kanji Furuya, Keita Aoki, Hironori Niki
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  ABSTRACT: Measuring relative genetic distances is one of the best ways to locate genetic loci. Here we report the construction of a strains set for genetic mapping in Schizosaccharomyces japonicus, which belongs to the genus Schizosaccharomyces together with the well-studied fission yeast Sz. pombe. We constructed 29 strains that bear a positive-negative selection marker at different loci. The marker was inserted every 500 kb in the genome of Sz. japonicus. Each marker thus becomes a 'scale mark' of a chromosome that behaves like a yardstick. By determining the genetic distances from the inserted markers, the relative location of a genomic mutation can be determined. We also constructed a fosmid library that covers an entire genome of Sz. japonicus. These tools together would facilitate identification and cloning of the gene.
  Yeast 05/2012; 29(6):241-9. · 1.89 Impact Factor
• Article: Genome-wide identification and characterization of replication origins by deep sequencing.

  Jia Xu, Yoshimi Yanagisawa, Alexander M Tsankov, Christopher Hart, Keita Aoki, Naveen Kommajosyula, Kathleen E Steinmann, James Bochicchio, Carsten Russ, Aviv Regev, Oliver J Rando, Chad Nusbaum, Hironori Niki, Patrice Milos, Zhiping Weng, Nicholas Rhind
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  ABSTRACT: DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood. We have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions. The sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.
  Genome biology 04/2012; 13(4):R27. · 6.63 Impact Factor
• Article: Breakage of the nuclear envelope by an extending mitotic nucleus occurs during anaphase in Schizosaccharomyces japonicus.

  Keita Aoki, Hanako Hayashi, Kanji Furuya, Mamiko Sato, Tomoko Takagi, Masako Osumi, Akatsuki Kimura, Hironori Niki
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  ABSTRACT: During open mitosis in higher eukaryotic cells, the nuclear envelope completely breaks down and then mitotic chromosomes are exposed in the cytoplasm. By contrast, mitosis in lower eukaryotes, including fungi, proceeds with the nucleus enclosed in an intact nuclear envelope. The mechanism of mitosis has been studied extensively in yeast, a closed mitosis organism. Here, we describe a form of mitosis in which the nuclear envelope is torn by elongation of the nucleus in the fission yeast Schizosaccharomyces japonicus. The mitotic nucleus of Sz. japonicus adopted a fusiform shape in anaphase, and its following extension caused separation. Finally, a tear in the nuclear envelope occurred in late anaphase. At the same time, a polarized-biased localization of nuclear pores was seen in the fusiform-shaped nuclear envelope, suggesting a compromise in the mechanical integrity of the lipid membrane. It has been known that nuclear membrane remains intact in some metazoan mitosis. We found that a similar tear of the nuclear envelope was also observed in late mitosis of the Caenorhabditis elegans embryo. These findings provide insight into the diversity of mitosis and the biological significance of breakdown of the nuclear envelope.
  Genes to Cells 07/2011; 16(9):911-26. · 2.68 Impact Factor
• Source

  Available from: Conrad Alexander Nieduszynski

  Article: Comparative functional genomics of the fission yeasts.

  Nicholas Rhind, Zehua Chen, Moran Yassour, Dawn A Thompson, Brian J Haas, Naomi Habib, Ilan Wapinski, Sushmita Roy, Michael F Lin, David I Heiman, [......], Hanah Margalit, Rob Martienssen, Conrad A Nieduszynski, Joseph W Spatafora, Nir Friedman, Jacob Z Dalgaard, Peter Baumann, Hironori Niki, Aviv Regev, Chad Nusbaum
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  ABSTRACT: The fission yeast clade--comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus--occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.
  Science 05/2011; 332(6032):930-6. · 31.20 Impact Factor
• Article: Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus.

  Keita Aoki, Reiko Nakajima, Kanji Furuya, Hironori Niki
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  ABSTRACT: Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4(+) of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early-log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4(+) probe. The plasmid containing ade6(+) suppressed the auxotrophic growth of the ade6-domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus.
  Yeast 12/2010; 27(12):1049-60. · 1.89 Impact Factor
• Article: [Regulation of mitosis and chromosome segregation: overview].

  Keita Aoki, Hironori Niki
  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 03/2009; 54(4 Suppl):406-8.
• Article: CDC2 phosphorylation of the fission yeast dis1 ensures accurate chromosome segregation.

  Keita Aoki, Yukinobu Nakaseko, Kazuhisa Kinoshita, Gohta Goshima, Mitsuhiro Yanagida
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  ABSTRACT: Shortened kinetochore microtubules take separated chromatids to the opposing spindle poles in anaphase. Fission yeast Dis1 belongs to the Dis1/XMAP215/TOG family that is required for proper microtubule dynamics. Here, we report that Dis1is regulated by Cdc2 phosphorylation and that this mitotic phosphorylation ensures the fidelity of chromosome segregation. Whereas mutants Dis1(6A) and Dis1(6E) that substitute all of the six Cdc2 sites for Ala or Glu, respectively, produce colonies at 22 degrees C-36 degrees C, Dis1(6A) but not Dis1(6E) loses a minichromosome and reveals aberrant chromosome segregation at significant frequencies. Dis1(WT) is recruited to two regions of the mitotic spindle: kinetochores (possibly also kinetochore microtubules) in metaphase and the pole-to-pole microtubule lattice in anaphase. Mutant Dis1(6E) preferentially binds to metaphase kinetochores, whereas Dis1(6A), which is located along microtubules, fails in its accumulation at kinetochores. Dis1(6A) displays synthetic lethality with the mis12-537, which is a mutant that compromises kinetochore function. Dis1(6E) mimics the Cdc2-phosphorylated form of Dis1(WT), whereas Dis1(6A) can partially rescue the phenotype resulting form deletion of Mtc1/Alp14, another XMAP215-like protein. In anaphase, dephosphorylated Dis1 and Dis1(6A), but not Dis1(6E), move to the spindle microtubule lattice near the SPBs. Cdc2 thus directly phosphorylates Dis1, and this phosphorylation regulates Dis1 localization in both metaphase and anaphase and ensures high-fidelity segregation.
  Current Biology 09/2006; 16(16):1627-35. · 9.65 Impact Factor
• Article: An interactive gene network for securin-separase, condensin, cohesin, Dis1/Mtc1 and histones constructed by mass transformation.

  Tatsuro Yuasa, Takeshi Hayashi, Nobuyasu Ikai, Toshiaki Katayama, Keita Aoki, Takayuki Obara, Yusuke Toyoda, Takeshi Maruyama, Daisuke Kitagawa, Kohta Takahashi, Koji Nagao, Yukinobu Nakaseko, Mitsuhiro Yanagida
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  ABSTRACT: 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.
  Genes to Cells 12/2004; 9(11):1069-82. · 2.68 Impact Factor