Abstracts

有中心粒太陽虫類におけるミトコンドリアゲノムの解読.

有中心粒太陽虫類は主に淡水環境で生息する従属栄養性の原生生物である。近年行われた核コード遺伝子に基づく大規模分子系統解析から、有中心粒太陽虫類はハプト藻類と近縁だと考えられている。核ゲノム情報が蓄積される一方、有中心粒太陽虫類のミトコンドリアゲノム(mtDNA)については未着手であったので、本研究は当分類群に属する未記載種SRT127とPolyplacocystis contractilisのmtDNAを解読した。両種のmtDNAはともに約110kbの環状ゲノムで、祖先のバクテリアに由来するタンパク質遺伝子を41種類もっていた。さらに転移性イントロンであるグループI(gI)イントロンがSRT127のmtDNAには19個、P. contractilisには10個含まれていた。gIイントロンの起源を探るために、イントロン内部にコードされているホーミングエンドヌクレアーゼを用いた系統解析や、イントロン配列の比較を行った。その結果、有中心粒太陽虫類のgIイントロンの一部は緑藻類の葉緑体ゲノム内のものと起源が同一であることが示された。このことは系統的に離れた生物の異なるオルガネラ間で、イントロンの水平伝播が起こったことを示唆している。また興味深いことに、解読した2種のmtDNAのうちP. contractilisでのみmRNAでC-to-U型RNAエディティングが起こることが確認された。2種のmtDNAや有中心粒太陽虫類のRNA-seqを比較しながら、当分類群においてC-to-U型RNAエディティングが成立した過程を考察する。

外洋性渦鞭毛藻Ornithocercus magnificusに見られる共生シアノバクテリアのゲノム解析.

Ornithocercus属の渦鞭毛藻は，著しく発達した横溝翼片によって作られる空間にシアノバクテリアを共生させる。このような形態的特徴から共生体と宿主渦鞭毛藻は強い共生関係を結んでいると予想されるが，Ornithocercus属渦鞭毛藻は難培養性のため，これまで限られたアプローチでしか研究が行われてこなかった。特に分子生物学的な研究は限られており，共生体および宿主ともにrDNA 配列など特定の分子情報が報告されているのみである。本研究ではOrnithocercus属渦鞭毛藻に見られる共生関係が，共生体の進化にどのような影響を与えたかを調べるため，渦鞭毛藻Ornithocercus magnificusの共生体ゲノムの解読を行なった。ゲノム増幅によって得られたO. magnificusの共生体DNAを解析した結果，総塩基数約1.88 Mbpのドラフトゲノムが得られた。近縁な自由生活性α-シアノバクテリアのゲノム情報との比較解析を行なったところ，共生体ゲノム上の遺伝子レパートリーには一定の縮退傾向が見られるものの，主要な代謝経路に関わる遺伝子はほぼ全て保存されており，宿主に対する依存の程度は大きくないことが予想された。本発表ではゲノム配列および遺伝子情報から予想される代謝的特徴に基づき，本共生体の進化について論じたい。

Mitochondrial genome of strain SRT308: insights into structure and gene content fo the ancestral euglenozoan mitochondrial genome.

Euglenozoa is a large protist assemblage comprising three major subgroups, namely Euglenida, Diplonemea and Kinetoplastea. Members of this assemblage are known for a large diversity in mitochondrial (mt) genome structure. The mt genome of Euglena gracilis, a representative of euglenids, are likely composed of multiple linear chromosomes, which harbor only 6 protein-coding genes and fragmented ribosomal RNA genes. No RNA editing has been detected in the transcripts from Euglena mt genome. Diplonemid mitochondrial genomes were found to comprise multiple minicircular chromosomes, each of which carries a partial gene fragment, and initial transcripts from the mt minicircles receive both trans-splicing and RNA editing to yield mature mRNAs. Mitochondria of members of Kinetoplastea contain a complex network of two types of circular chromosomes, maxicircles and minicircles, and mt gene transcripts are edited intensively prior to translation. To understand how the complex genome structures and RNA editing mechanisms observed among the extant euglenozoans emerged, it is important to infer the mt genome structure of the ancestral euglenozoan. The aforementioned issue can be addressed by analyzing the mt genome of a novel protist strain SRT308, which was placed robustly at the base of the Euglenozoan clade in our phylogenomic analysis based on 153 nucleus-encoded gene sequences. We successfully reconstructed the complete SRT308 mt genome of 61 Kb in length in this study. In this mt genome, we identified 35 open reading frames (ORFs) and 29 structural RNA genes (2 and 27 are rRNA and tRNA genes, respectively). 19 out of the 35 ORFs are functionally assignable, and some of them were not found in Euglena, Diplonema or kinetoplastids. The SRT308 mt genome suggests that this protist (and the ancestral euglenozoan as well) possesses the mt genome larger in both size and gene content than the euglenozoan mt genomes studied to date.

Genome analysis of a symbiotic cyanobacterium in a dinophysialean dinoflagellate, Orthocercus magnificus.

Many dinoflagellate species belonging to Dinophysiales are known to possess cyanobacterial symbionts. Members of a dinophysialean genus Ornithocercus arrange characteristic chambers enclosed by developed cingular lists for their symbionts, implying that this partnership is not trivial for both host and symbiont. To our knowledge, there is no laboratory culture of any dinophysealeans harboring cyanobacterial symbiont, and the dinoflagellate-cyanobacterium interaction has been studied mainly by microscopic observations on the cells isolated from sea water samples. To obtain deeper insights into the cyanobacterial symbiosis in dinophysealeans, we amplified and sequenced the genome of symbiotic cyanobacteria isolated from a single O. magnificus cell. By comparing to the genomes of free-living cyanobacteria, we detected a reductive trend in the genome of the O. magnificus symbiont, which is likely an outcome from the adaptation to a symbiotic lifestyle. Nevertheless, the magnitude of gene loss appeared to be less severe in the genome of the O. magnificus symbiont than those of other cyanobacterial symbionts, suggesting the symbiont dependents metabolically on the host at a relatively low level. The genome data from the cyanobacterial symbiont in O. magnificus is consistent with the hypothesis of the host growing cyanobacteria in the chamber as preys.

Comparative genomics of photosynthetic and non-photosynthetic Cryptomonas (Cryptophyta) species.

The loss of photosynthesis has occurred multiple times in eukaryotic lineages and the remaining non-photosynthetic plastids take essential roles other than photosynthesis such as fatty acid, isoprenoid, and amino acid biosyntheses. The unicellular algal genus Cryptomonas (Cryptophyta) contains both photosynthetic and non-photosynthetic members, the latter having lost the photsynthetic ability on at least three separate occasions. In order to elucidate the evolutionary process underlying the loss of photosynthesis in Cryptomonas, we sequenced the plastid genomes of two non-photosynthetic strains, Cryptomonas sp. CCAC1634B and SAG977-2f, and compared them to the previously sequenced plastid genome of the non-photosynthetic C. paramecium CCAP977/2a. Also, the plastid genome of photosynthetic Cryptomonas curvata CCAP979/52 was sequenced as reference. The genome sizes of non-photosynthetic plastids are 106,661 bp, 80,503 bp and 77,717 bp in SAG977-2f, CCAC1634B and CCAP977/2a, respectively. Although the most of photosynthesis related genes such as psa and psb gene families were disappeared from the non-photosynthetic plastid genomes, a few pseudogenes retained in SAG977-2f. While the gene order of the photosynthetic plastids is roughly common among Cryptophyte genera, the genome recombinations are seen in the smaller genomes of the non-photosynthetic plastid genomes more frequently. Intriguingly, the light-independent protochlorophyllide reductase comprising of chlB, L and N retain in SAG977-2f, CCAC1634B despite the loss of photosynthesis. On the other hand, while CCAP977/2a maintain rubisco related genes including rbcL, rbcS and cbbX, the other two non-photosynthetic strains lost the rubisco proteins. In addition, we present the draft nuclear genome sequences of two non-photosynthetic Cryptomonas strains, CCAC 1634B and CCAP977/2a. We discuss the evolutionally mechanisms of loss of photosynthesis and the divergence of non-photosynthetic plastids in Cryptomonas.

Novel eukaryotes for elucidating the early eukaryotic evolution.

One of the most fundamental questions in biology is how modern eukaryotic lineages have been diversified from the last eukaryotic common ancestor. Recent large-scale multigene (phylogenomic) studies on diverse eukaryotes clarified local branching patterns in the tree of eukaryotes, but we have yet to resolve the relationship amongst early diverged branches with confidence. There are two major obstacles to reconstruct the deep eukaryotic branches with high confidence, namely limitations on the size and taxon-sampling of data matrices for phylogeny. Firstly, phylogenetic signal in the data matrices analyzed in recent phylogenomic studies is likely insufficient to resolve the deep nodes of the tree. With respect to rapid growth of sequence data, this issue can be overcome in the near future by generating and analyzing data matrices larger than the previously analyzed ones. Secondly, the data matrices analyzed to date may have missed key taxa/lineages that are critical to resolve early eukaryotic evolution. The second issue potentially demands much more effort than the first one, as we need to discover novel eukaryotes in natural environments. I and my colleagues have been surveying, isolating and cultivating potentially novel eukaryotes for years. In this presentation, I introduce a series of novel “deep-branching” eukaryotes, of which we isolated and established laboratory cultures. We also discuss the significance of the mitochondrial or plastid genomes of some of those eukaryotes in the context of organellar genome evolution. Well, I should stop working on this abstract, since I’m currently in an ocean-view hotel room in Langkawi, Malaysia.

Phylogenomic approach to the early evolution of Foraminifera.

Foraminifers use diverse types of materials for their tests, namely hyaline calcareous, porcelaneous calcareous, agglutinated, and pseudochitinous. Such diversity in test-material has not been found in any group of eukaryotes except foraminifers, and prompts many paleontological studies on the foraminiferal evolution. For the order-level foraminiferal phylogey, BouDagher-Fadel (2008) proposed Allogromida with pseudochitinous tests as the most basal branching group prior to the separation of Taxtulariida with agglutinated tests and Milliolida with porcelaneous calcareous tests. However, this hypothesis based on fossil records remains the position of Rotaliida with hyaline calcite tests unclear in the foraminiferal tree. Noteworthy, a potentially serious problem in fossil record-based studies is that water-soluble or fragile tests could have been lost, causing sporadic fossil records of particular foraminifers. An alternative scenario for the foraminferal phylogeny was proposed based on a molecular phylogeny of small subunit rRNA sequences (Pawlowski et al., 2013). In this scenario, the ancestral foraminiferal group, Alloromida, was diverged into two independent agglutinated groups (Textulariida and Spirillinida), and Milliolida and Rotallida were then emerged separately from Textilariida and Spirillinida, respectively. Unfortunately, the rRNA phylogeny failed to resolve the relationship among the aforementioned orders in foraminifers with high statistical support. Thus, we need a phylogenomic dataset to elucidate the relationship among major groups in foraminifers with confidence. In this study, we generated and analyzed a 125-gene dataset covering representative species of each of Taxtulariida, Spirillinida, Milliolida and Rotaliida, together with the genome data from Retisulomyxa filosa (Allogromida). Our phylogenomic analysis provides the concrete ground to discuss the unresolved issues in early foraminiferal evolution.

オピストコンタおよび近縁系統におけるオートファジー関連遺伝子の網羅的探索.

オートファジーは細胞内物質循環に関わる機構であり、真核生物の広い系統で保存されていると考えられている。しかし、モデル生物および人獣病原性の生物以外のオートファジー機構に関する情報は十分とは言えず、真核生物全体でオートファジー機構がどれほど普遍的に保存されているかは検証が必要である。そこで、本研究ではオートファジー研究の最も進んだ酵母・ヒトを含むオピストコンタおよびその近縁系統（Apudomonadida、Ancyromonadida、Breviatea）を対象に、オートファジー機構の普遍性を検証した。オピストコンタおよびその近縁系統の多様性を考慮した32生物種を対象とし、トランスクリプトームデータに対してアミノ酸配列相同性に基づきATGとその関連遺伝子の候補配列を検索した。その結果、微胞子虫を除く全てのオピストコンタおよび近縁系統からオートファジーに必須とされる遺伝子ATG8とその関連遺伝子を検出した。従って、オートファジー機構はオピストコンタとその近縁系統が分岐する以前に確立されていたと予想できる。微胞子虫類はRozella allomycesに代表される菌類の最原始系統から分岐したのち、オートファジー機構を二次的に失ったと考えられる。

渦鞭毛藻細胞内共生珪藻にもオートファジーはあるのか?!

オートファジーは真核細胞内でのタンパク質のリサイクルを担う普遍的分子機構と考えられるが、その研究は酵母や一部のモデル生物等に限定されている。ゆえに、多くの原生生物においても、その実態は解明されていない。また、渦鞭毛藻細胞内共生珪藻は葉緑体のみならず、核やミトコンドリアをも保持しており、縮退が進んでいない共生体として知られる。さらに共生珪藻のオートファジーが残存すれば、宿主と共生体の両者にオートファジーが保存されている初の知見となる。本研究ではその実態解明の初手として、珪藻と渦鞭毛藻RNA-seqデータからオートファジー関連遺伝子プロファイルを作成し、共生珪藻を保持する渦鞭毛藻2種のデータ中にオートファジー関連遺伝子を探索した。その結果、2種の渦鞭毛藻データ中から宿主と共生体のオートファジー関連遺伝子が検出されたため、共生珪藻はオートファジー系を保持していることが強く示唆された。

有殻アメーバのゲノム解析から見えてきた一次細胞内共生進化におけるDNAウイルスの役割

有殻アメーバ Paulinella microporaは、植物の葉緑体とは起源が異なるシアノバクテリア由来のオルガネラを持つユニークな光合成生物であり、一次細胞内共生進化の初期過程を解明する上で鍵となる生物と考えられている。我々はP. microporaのゲノム解析を行い、同生物のゲノムに新種の DNA ウイルスの配列が大量に挿入されていることを発見した。驚いたことに、有殻アメーバゲノムに転移したウイルス由来の遺伝子について解析をしたところ、細胞内共生が始まって間もない、大規模な遺伝子転移が生じた時期に、ウイルスの感染が始まったことが示唆され、一次共生進化に DNA ウイルスが関わることが示唆された。

Paulinella micropora is a thecate amoeba possessing a cyanobacteria-derived organelle, termed as chromatophore. Since the chromatophore was acquired more recently than the chloroplasts of plants, P. micropora is considered as a model organism to study the initial phase of primary endosymbiotic evolution. We analyzed P. micropora's genome and found that novel DNA viruses have infected in this organism during the evolution. Interestingly, it was estimated that the virus's infections started at the age when the massive gene transfer occurred in P. micropora. We discuss the role of DNA viruses in the gene transfer of the primary endosymbiosis.