Abstract

Residual nuclear genome of the green algal endosymbiont in the dinoflagellate Lepidodinium chorophorum.

Cryptophytes and Chlorarachnionphytes are unique in possessing two nuclei in their cells. In these 'complex' algal cells, the conspicuous nucleus in the cytosol is of the host cell. On the other hand, the second, highly-reduced nucleus, nucleomorph, is found in the space corresponding to the cytosol of the endosymbiotic alga (periplatidal compartment or PPC). The nucleomorphs, are most likely of the endosymbiotic algae, and anticipated to retain the key information regarding the reductive genome evolution during the endosymbiont-to-organelle transition. Lepidodinum chlorophorum is a member of dinoflagellates, and highly likely experienced plastid replacement involved in a green alga. Early microscopic works identified a nucleus-like structure in the PPC, but no molecular sequence for the nucleomorph in Lepidodinium was reported. In this study, we successfully isolated two sets of eukaryotic small and large subunits of ribosomal RNA (SSU and LSU rRNA) sequences from L. chlorophorum. The first set of SSU and LSU rRNA sequences was likely amplified from the dinoflagellate nuclear genome. The second, divergent rRNA set appeared to bear only partial similarity to the homologues. We experimentally confirmed that these SSU and LSU rRNA sequences were next to each other, albeit no 5.8S rRNA sequence was detected in the intergenic region. Significantly, our LSU phylogeny recovered a robust affinity between the second LSU rRNA sequence of L. chlorophorum and green algae, suggesting that these rRNA sequences were derived from the nuclear genome of the green alga that gave rise to the current Lepidodinium plastids.

Trans-splicing in the intron-poor eukaryotic parasite Giardia intestinalis.

Genome reduction most likely occurred in the transition process from a free living organism to a parasite, and it remains elusive how this process alters the organization and expression of genes encoded in the corresponding genome. In this study, we revealed that a highly reductive nuclear genome of the intestinal parasite against human and animals Giardia intestinalis possesses an exclusively unique gene expression system. Some protein-coding genes in Giardia are split into several pieces and transcribed independently. Two particular pre-mRNAs directly interact with each other by forming an intermolecular-stem structure and trans-spliced into a mature mRNA by spliceosomes.

Molecular evidence for the residual nuclear genome of the green algal endosymbiont in the dinoflagellate Lepidodinium chlorophorum.

Members of the dinoflagellate genus Lepidodinium acquired the current plastids from an endosymbiotic green alga. Microscopic works identified a residual nucleus, “nucleomorph,” in the space corresponding to the cytosol of the endosymbiotic green alga, but no molecular evidence has yet to be reported. In this study, we provide the first molecular evidence for the nucleomorph in Lepidodinium chlorophorum. The nucleomorph genome in Lepidodinium, by combining the genome sequences of the nucleomophs in cryptophytes and chlorarachniophyes, anticipated to provide key information regarding the reductive genome evolution during the transition from a endosymbiont to an organelle fully integrated into the host cell.

Giardia intestinalisゲノム中の「分割」イントロン群

Giardia intestinalisのドラフトゲノム解析から、本種の遺伝子レパートリーやゲノム構造は、他の真核生物と比べてシンプルであると信じられている。特に、真核ゲノムに普遍的に存在するスプライソゾーマルイントロンは、これまでGiardiaドラフトゲノム中に4個しか同定されていない。しかし、我々はGiardiaの90 kDa熱ショックタンパク質遺伝子（hsp90）中に、「分割」イントロンを発見したので報告する。

Giardia Hsp90タンパクのN末・C末部分は、ゲノム中で互いに約0.8 Mbp離れた領域にコードされ、それぞれRNAへと転写される。しかし我々は、さらに全コード領域を含むmRNAを検出した。上述3種類のRNA配列比較から、N末RNAの3' 非翻訳領域（UTR）はGUで始まり、C末RNAの5' UTRはGiardiaイントロン間で高度に保存された配列モチーフを含みAGで終わることが分かった。また、N末RNAの3'UTRとC末RNAの5' UTRはステム構造を形成しうる。つまり2つの未成熟RNAはステム構造を介してイントロンを形成し、transスプライシングを通じて成熟mRNAが完成すると考えられる。同様の分割イントロンはhsp90以外の2遺伝子中にも存在する。これら分割イントロン群はGiardiaゲノムにおけるイントロン進化を理解する上で重要な発見となる。

Molecular evidence for the residual nuclear genome of the green algal endosymbiont in the dinoflagellateLepidodinium chlorophorum.

Cryptophytes and Chlorarachnionphytes acquired their plastids through endosymbiotic red and green algae, respectively. What the two algal lineages makes unique is that these cells possess two nuclei. In these 'complex' algal cells, the conspicuous nucleus in the cytosol is of the host cell. On the other hand, the second, highly-reduced nucleus is found in the space corresponding to the cytosol of the endosymbiotic alga (so-called the periplatidal compartment or PPC). The residual nuclei in the PPC in cryptophytes and chlorarachniophytes, nucleomorph, are most likely of the endosymbiotic algae, and anticipated to retain the key information regarding the reductive genome evolution during the endosymbiont-to-organelle transition.

Lepidodinum chlorophorum and L. viride are members of dinoflagellates. Although typical photosynthetic dinoflagellates utilize the plastids containing chlorophyll c and a unique carotenoid peridinin, Lepidodinium acquired the current plastids from a green algal endosymbiont. Early microscopic works identified a nucleus-like structure in the PPC of Lepidodinium cells. Nevertheless, these micrographs are, to our knowledge, the sole evidence for the nucleomorph in the Lepidodinium cells.

In this study, we successfully isolated two sets of eukaryotic small and large subunits of ribosomal RNA (SSU and LSU rRNA) sequences from L. chlorophorum. Based on sequence similarity, we concluded that the first set of SSU and LSU rRNA sequences was amplified from the dinoflagellate nuclear genome. The second rRNA set, particularly SSU rRNA sequence, appeared to be extremely divergent and bear only partial similarity to the homologues in GenBank database. We experimentally confirmed that these SSU and LSU rRNA sequences were next to each other, albeit no 5.8S rRNA sequence was detected in the intergenic region. Although the second SSU rRNA sequence is too divergent for phylogenetic analysis, the second LSU rRNA sequence retains a reasonable sequence similarity to the homologues from other eukaryotes. Significantly, our LSU phylogeny recovered a robust affinity between the second LSU rRNA sequence of L. chlorophorum and green algae, suggesting that these rRNA sequences were derived from the nuclear genome of the green alga that gave rise to the current Lepidodinium plastids. Consequently, this work provides the first molecular evidence for the nucleomorph in this dinoflagellate species.

Evolutionary characteristics of the green-colored plastid in the dinoflagellate Lepidodinium chlorophorum.

Most photosynthetic dinoflagellates possess plastids containing chlorophyls a+c, but species belonging to the genus Lepidodinium are unique in bearing non-canonical plastids containing chlorophylls a+b. According to the pioneering works on pigment composition data, it has been proposed that Lepidodinium plastids were derived from a prasinophyte species, though this hypothesis was not supported by a recent phylogenetic analysis based on an alignment comprised of eight plastid proteins (Takishita et al. 2008). This "8-protein" analysis however was insufficient to clarify the origin of Lepidodinium plastids for two major reasons: First, the alignment lacked sufficient evolutionary information to resolve the precise origin of Lepidodinium plastids. Second, the taxa considered did not well represent the diversity of Chlorophyta. Particularly, prasinophytes were poorly sampled in the alignment. In this study, we sequenced plastid-encoded genes from L. chlorophorum, one pedinophyte species, one ulvophyte species, and six prasinophyte species. The 85 sequences newly determined in this study and recent progress in plastid genome sequencing enabled us to prepare an alignment comprised of 11 plastid proteins from green algal taxa that appropriately cover the diversity of Chlorophyta. All the analyses of the 11-protein data set robustly grouped L. chlorophorum with members of the "core chlorophytes." We additionally analyzed the pigment composition in L. chlorophorum, and found that the suit of the pigments in this dinoflagellate apparently lacked prasinoxanthin. Considering the results from the analyses described above, we propose that Lepidodinium plastids are of core chlorophyte in origin. Finally, we discuss the deviant genetic code used in Lepidodinium plastid genome.

Spliceosome-mediated trans-splicing produces the complete mRNA for heat shock protein 90 in Giardia intestinalis.

The diplomonad Giardia intestinalis is a causative agent of giardiais. G. intestinalis is extremely unique in the perspective of intron evolution in eukaryotes, since a handful of short spliceosomal introns have been identified in its complete nuclear genome, and the components of spliceosomes may be highly reduced or divergent in this organism. In G. intestinalis, all known intron-containing transcripts are thought to undergo cis-splicing, but we revealed that G. intestinalis employs a novel type of trans-splicing to generate the mature transcripts of the gene encoding heat shock protein 90 (HSP90).

In chromosome X, the N and C termini of HSP90 are encoded in two distinctive regions separated by ~0.8 Mbp, and no other HSP90-like open reading frame was detected in the genome data. On the other hand, we identified three kinds of hsp90 transcripts with poly-A tail at the 3' end, the major type covering both N- and C-termini, while two minor types covering the N- or C-terminus. Intriguingly, the 3' untranslated region of the N-terminal transcript starts with GU, and the 5' untranslated region of the C-terminal transcript includes a branch point-like sequence and ends with AG, being consistent with the previously-known spliceosomal introns in G. intestinalis. These finding prompted us to speculate that the complete hsp90 transcripts are produced by the N- and C-terminal transcripts via spliceosome-mediated trans-splicing. Our hypothesis is strongly supported by an experimental evidence for a Y-shaped molecule produced by a 5'-2' link between GU of the 'left intron-piece' and the branch point sequence in the 'right intron-piece,' which corresponds to the lariat structure from canonical spliceosomal introns. This trans-splicing is most likely facilitated by physical interaction between the two pre-mature transcripts via a 26 bp-long stem structure formed between the two intron-pieces.

Green-colored plastids in the dinoflagellate genus Lepidodinium are of core chlorophyte origin.

Most photosynthetic dinoflagellates possess plastids containing chlorophyls a+c, but species belonging to the genus Lepidodinium are unique in bearing non-canonical plastids containing chlorophyls a+b. According to the pioneering works on pigment composition data, it has been proposed that Lepidodinium plastids were derived from a prasinophyte species, though this hypothesis was not supported by a recent phylogenetic analysis based on an alignment comprised of eight plastid proteins (Takishita et al. 2008). This "8-protein" analysis however was insufficient to clarify the origin of Lepidodinium plastids for two major reasons: First, the alignment lacked sufficient evolutionary information to resolve the precise origin of Lepidodinium plastids. Second, the taxa considered did not well represent the diversity of Chlorophyta. Particularly, prasinophytes were poorly sampled in the alignment. In this study, we sequenced 85 plastid-encoded genes in total from L. chlorophorum, one pedinophyte species, one ulvophyte species, and six prasinophyte species. These new sequence data and recent progress in plastid genome sequencing enabled us to prepare an alignment comprised of 11 plastid proteins from green algal taxa that appropriately cover the diversity of Chlorophyta. All the analyses of the 11-protein data set robustly grouped L. chlorophorum with members of the "core chlorophytes" Thus, we here propose that Lepidodinium plastids are of core chlorophyte in origin.

ストラメノパイル類における翻訳伸長因子遺伝子の進化

細胞内におけるタンパク質合成の中心的役割を果たすことが知られている翻訳伸長因子タンパク質(EF)は，真核生物では特にeEF-1α と呼ばれる。eEF-1α を有する真核生物に加えて，eEF-1α を持たず翻訳伸長因子様遺伝子(EFL)を有する生物も近年報告され，真核生物におけるeEF-1α/EFL進化が注目されている。これまでの知見からは，真核生物の共通祖先はeEF-1α遺伝子のみを有し，真核生物進化の過程においてeEF-1 α遺伝子が受け継がれ(vertical gene transfer, VGT) ，その後eEF-1α 遺伝子から進化したEFL遺伝子が遺伝子水平伝播(lateral gene transfer, LGT)によって広がったと考えられている。

本研究ではストラメノパイル類のEF遺伝子探索を行い，分子系統解析結果に基づきストラメノパイル類eEF-1α/EFL進化について考察した。遺伝子探索の結果，珪藻類とラン菌類Pythium属からEFL遺伝子が検出されたが，本生物以外からはeEF-1α 遺伝子のみを検出した。さらに珪藻類およびPythium属の中にはEFL遺伝子とeEF-1α 遺伝子を両方有する種も発見された。分子系統解析の結果，珪藻類とPythium属EFLの姉妹群関係が強く支持された一方，珪藻類とPythium属eEF-1α は他のストラメノパイル類からのeEF-1α に近縁であることが判明した。従って珪藻類，ラン菌類のEFL遺伝子進化に関し、以下3つの可能性が考えられる：(1)本生物共通祖先からのEFL/eEF-1α のVGT，(2)同一もしくは近縁なドナーからの独立なEFL遺伝子LGT，(3)珪藻―ラン菌間におけるEFLのLGT。

配列組成の極端な変化によるモデル不整合が分子系統解析に与える影響について

分子系統解析においてOTU間で極端に塩基あるいはアミノ酸組成が異なる場合、置換モデルと実配列の経験した置換パターンとの間に不整合が起こりアーティファクトが誘導される可能性が示唆されていた。しかし、真の系統樹が未知である既存の生物種由来の配列を用いた解析では、推定結果がアーティファクトであると断定することが出来ない。また、組成の偏り以外の要因がアーティファクト誘導に影響している可能性を完全に排除できない。よって、配列間での組成の偏りがアーティファクトを誘導するのか、それがどの程度深刻な問題であるかについては詳細な検討がなされていない。

本研究では、OTU間の組成に偏りのある配列をモンテカルロシミュレーションを用いて生成し解析した。この解析では真の系統樹は予め分かっているため、推定結果が組成の偏りに起因するアーティファクトであるか否かを厳密に検証出来る。

一連の解析結果から、配列間での組成の偏りがアーティファクトを強く誘導すること、そのアーティファクトはデータサイズの拡大や配列数の増加だけでは軽減出来ないことなどが判明した。以上の結果は、実際の系統解析においても、例えば他の生物由来の相同配列とアミノ酸組成が極端に異なるApicoplast遺伝子配列を含む系統解析や、生物種間でGC含量に差があるSSUrRNA配列を用いた系統解析などでは同様の問題が起こっている可能性を示唆している。

クロロフィルa, bを持つ緑色渦鞭毛藻類Lepidodinium chlorophorumの葉緑体起源

光合成渦鞭毛藻の多くは、chlorophyll cを持ち、カロテノイド系色素としてペリディニンを持つ葉緑体を保持している。渦鞭毛藻類の祖先型葉緑体は、ペリディニンタイプであり、二次共生した紅藻類に由来すると考えられている。渦鞭毛藻類の中には、祖先型葉緑体を一度手放した後、三次共生により新たな葉緑体を獲得した生物種も知られている。渦鞭毛藻Lepidodinium chlorophorumは、祖先型葉緑体ではなく、chlorophyll bを含む緑色の葉緑体を保持している。L. chlorophorum葉緑体からプラシノ藻特有の色素を検出したとの報告もあり、この渦鞭毛藻類葉緑体はプラシノ藻起源であると考えられている。しかし、８種類の葉緑体遺伝子配列に基づく分子系統解析では、L. chlorophorum葉緑体はプラシノ藻類との近縁性を示さなかった。今回、６種のプラシノ藻類と２種の緑藻類から、新たに１１種類の葉緑体遺伝子配列を決定し分子系統解析を行い、三次共生植物L. chlorophorum葉緑体の起源を詳細に検討したので報告する。