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General Sessions (1A-01–3E-13) 304 Genes Genet. Syst. (2014) 89 General sessions (1A-01 – 3E-13) 1A NAGAKI, Kiyotaka1, TANAKA, Keisuke2, 1A ADACHI, Shun1,2, MURAKAWA, Yasuhiro1,3, -01 MATSUSHIMA, Ryo1, KOBAYASHI, Hisato2, -03 HIRAGA, Sota1 (1Dept. Radiation Genet., Grad. Sch. MURATA, Minoru1 (1Inst. Plant Sci. and Resources, Med., Kyoto Univ., 2Medical Research Project, Inst. Okayama Univ., 2NODAI Genome Res. Center, Tokyo Health BioSci., The Univ. Tokushima Grad. Sch., Univ. Agr.) 3Max Delbruck Center for Molecular Medicine Berlin- Buch) Development of a novel method for identifying centromeric DNA sequences Involvement of SecA, SecY and AcpP in chromosome partition of Escherichia coli ChIP using anti-CENH3 antibodies is a powerful tool for isolation of centromeric DNAs. However, ChIP requires high quality We have found evidence that SecA (secretory), SecY and AcpP antibodies, and it is sometimes difficult to raise high quality (acyl carrier protein) are involved in chromosomal partition of E. antibodies, which is an obstacle for the isolation. Here, we report coli, acting together with MukBEF, DNA gyrase and Topo IV. a novel method for the isolation, based on the fact that the sizes When each protein was inactivated, cells exhibited the partition between canonical and centromeric nucleosomes are different. In minus phenotype. Inactivation of SecA caused reduction of the maize and rice, shorter DNA fragments on the CENH3 plasmid superhelicity and increase of plasmid concatemers. This nucleosomes than those on the canonical H3 nucleosomes have effect closely resembled the effect observed under inactivation of been reported. To know its universality, we isolated chromatins both MukB and DNA gyrase. SecA, SecY and AcpP localized as from rice, tobacco and welsh onion, and digested them into mono- helical structures with condensed parts. Time-lapse experiments nucleosome sizes. The digested DNA was separated using agarose of GFP-fused proteins showed that the condensed parts of protein gels. Monomeric size and shorter DNA fragments were purified, moved for a short time within the cell. The dynamic localization and used as templates of qPCR. In the qPCR, centromeric DNAs of these proteins was inhibited by inactivation of each protein. were significantly concentrated in the shorter fractions. In The addition of 1 mM sodium azide, which inhibited the ATPase addition, we found the shorter fragments enrichment with the activity of SecA, perturbed immediately the dynamic localization centromeric DNA sequences by sequence-length comparison after of SecY-GYP. The addition of 1 mM sodium azide resulted in Illumina sequencing. These results imply shorter DNA fragments abnormal subcellular localization of MukB, SeqA (sequestration) on centromeric nucleosomes are a universal feature, which must and oriC DNA. The novel chromosome partitioning mechanism be useful for identifying centromeric DNAs without anti-CENH3 that is performed by SecA, SecY and AcpP presumably acts as a antibodies. ‘molecular tether’ between chromosome and inner cell membrane. 1A MURATA, Minoru1, KANATANI, Asaka1, 1A OHNO, Yuko1, ISHII, Kojiro1,2 (1Grad. Sch. Front. -02 KASHIHARA, Kazunari1, HIRONAKA, Akiko1, -04 BioSci., Osaka Univ., 2Institute for Academic NAGAKI, Kiyotaka1 (1Group of Nuclear Genomics, Initiatives, Osaka Univ.) Inst. Plant Sci. and Resources, Okayama Univ.) Immediate response to chromosome aneuploidy Generation of a stable 1-Mb-sized artificial ring chromosome “Euploidy” represents a complete set of chromosomes which bear in Arabidopsis an entire gene set, or the genome, and therefore becomes a Plant artificial chromosomes (PACs) have been anticipated as prerequisite for every cellular program. However, equal segrega- new vectors for the development of new crops. Recently, we have tion and perfect inheritance of the chromosome set is an developed a novel method for generating PACs in the model plant exhaustive labor and accompanied with a considerable risk of Arabidopsis thaliana using the Cre/LoxP and Ac/Ds systems. The failure at every cell division. It remains enigmatic why generated PAC, “AtARC1 (A. thaliana artificial ring chromosome eukaryotes should persist in carrying the genome in separate 1)”, contains only a short centromere domain consisting of 180-bp molecules for euploidy establishment. We have prepared and repeats approximately 250 kb in length, and its flanking 2.6-Mb analyzed the chromosomally-engineered fission yeast in which region of chromosome 2 long arm. AtARC1 is stable during centromere region of a given chromosome can specifically be mitosis in the absence of selection and transmissible to the next removed and thereby acentric chromosomes can be conditionally generation through meiosis. No phenotypic abnormalities ap- induced. We found that such conditional acentrics are mostly peared in plants when an AtARC1 was added to the wild-type. missegregated into the daughter cells, rather than being lost However, because AtARC1 contains approximately 150 protein- immediately. This chromosome-specific missegregation allows a coding genes that derived from the long arm of chromosome 2, the temporal expansion and accumulation of aneuploid cells in the dosage effects are not negligible. In this study, therefore, smaller growing culture. Importantly, these conditional aneuploids are artificial ring chromosomes were attempted to be generated as made freshly. We revealed a specific response associated with the reported previously for AtARC1. As a result, a 1-Mb-sized ring immediate aneuploid cells. We will discuss the causes and chromosome was successfully generated. This AtARC2 was also consequences of such an immediate cellular response to chromo- stable during mitosis and transmissible to the next generation, so some aneuploidy. it is more suitable as a chromosome vector than AtARC1. Genes Genet. Syst. (2014) 89 305 1A TSUNEWAKI, Koichiro1,MORI,Naoki2, 1A HIRANO, Hiro-Yuki1, SATO, Dai-Suke1 (1Dept. Biol -05 YOTSUMOTO, Tatsuya2, TAKUMI, Shigeo2 (1Kyoto -07 Sci., Grad. Sch. Sci., Univ. Tokyo) Univ. (Emeritus), 2Lab. Plant Genet., Grad. Sch. Agr. Sci., Kobe Univ.s, Kobe Univ.) Genetic interaction among genes responsible for spikelet morphology in rice Genetic effect of the Aegilops caudata plasmon on manifes- Plants in the grass family have a unique inflorescence unit, called tation of the Ae. cylindrica genome a spikelet. In general, the spikelet consists of several florets and a We are carrying a genetic research to examine genetic autonomy pair of glumes. The floret contains the floral organs, including a of the plasmon from the co-existing genome by reconstructing an pistil, stamens and lodicules, enclosed by the lemma and palea. Aegilops caudata plant from its own genome (CC) and the Unlike general grass spikelets, rice spikelets has only one floret plasmon that passed half of a century in common wheat (genome and the glumes are highly degenerated. Therefore, the lemma AABBDD). For its reconstruction, it became necessary to transfer and plea are most prominent spikelet organs in rice. This rice the caudata plasmon from the alloplasmic wheat with this spikelet structure enables us to find many mutants that have plasmon to Ae. cylindrica (CCDD) for bridging the plasmon abnormal morphology of spikelet organs, such as the lemma, transfer from the 6x wheat to 2x Ae. caudata. For this purpose, palea and sterile lemmas. The sterile lemma is a specific organ we bred an alloplasmic line of Ae. cylindrica,(caudata)-CCDD, by that is found in rice, but not in other grasses. We analyzed three transferring the caudata plasmon in the 50th backcross gener- mutants such as g1, eg1 and th1 that have defects in spikelet ation of the alloplasmic wheat, (caudata)-AABBDD, to Ae. morphology. The sterile lemma was homeotically transformed cylindrica. We observed 13 characteristics for 5 generations into the lemma in the g1mutant, and extra lemma was produced during its breeding, comparing those characteristics in each in the eg1 mutant. In the th1mutant, the widths of the lemma generation with those of its euplasmic line. They differed only in and plea were reduced. We generated double mutant of g1 th1 one point, namely, male semi-sterility expression in the (cauda- and eg1 th1. The th1 mutation affected the extra lemma of eg1as ta)-CCDD. Comparison of 44 SSR loci in two organellar genomes seen in the original lemma, whereas it did not affect the between the alloplasmic and euplasmic Ae. cylindrica lines transformed lemma of g1. This difference may be related to the revealed 16 chloroplast and 6 mitochondrial SSR loci differing origin of the two ectopic lemmas. between them. 1A HONMA, Yujiro1, TAGUCHI, Kazunori2, HIYAMA, 1A NASUDA, Shuhei1, WATANABE, Shota1, IGARASHI, -06 Hajime1, YUI-KURINO, Rika1, HAMADA, Hiroyuki1, -08 Taro1, YOSHIOKA, Motohiro1, TSUJIMOTO, KUBO, Tomohiko1 (1Grad. Sch. Agr., Hokkaido U., Hisashi2, ENDO, Takashi R.1 (1Lab. Plant Genet., 2NARO, Hokkaido Agricultural Research Center) Grad. Sch. Agr., Kyoto Univ., 2Arid Land Research Center, Tottori Univ.) Characterization of candidate genes for restorer-of-fertility 2 of cytoplamsic male sterility in sugar beet (Beta vulgaris L.) Radiation hybrid mapping of the genes related to the gametocidal action in wheat Cytoplasmic male sterility (CMS) is maternally inherited male sterility. Plant CMS is associetd with mitochondrial ORF specific Several species of the genus Aegilops, wild relatives of wheat, to CMS plant. However, CMS expression suppressed by nuclear carry gametocidal (Gc) genes. The Gc gene Gc3-C1 derived from
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