コムギ染色体欠損系統を用いた新規活性型レトロトランスポゾン TriRe-1 の分子遺伝学的解析

　Retrotransposons constitute the large fraction (～80%) of the wheat genome where numerous and diverse retrotransposon families exist, where especially the long terminal repeat (LTR) retrotransposon family is known to be predominant. Thus, they have been considered to contribute to the genome expansion, sequence diversification and the genome structure alternation in the wheat genome. In addition, the insertion polymorphism of the LTR retrotransposon family among the cultivars has been known to be quite useful for the genetic analysis such as the linkage mapping and the phylogenetic studies. Here, we report the characteristics of a novel active LTR retrotransposon family TriRe‒1, which belongs to the Ty1‒copia group in the hexaploid wheat (Triticum aestivum L.) genome. This retroelement appears to encode all proteins required for the transposition and showed high insertion polymorphism among the hexaploid wheat cultivars, suggesting its potential of transpositional activity with at least recent transposition during wheat evolution. We studied the chromosomal localization of the TriRe‒1 insertion site based on the genome-wide comparative analysis using the nullisomic-tetrasomic lines of the cultivar Chinese Spring. The results showed that although the majority of the TriRe‒1 insertion sites exist across the homoeologous chromosomes of A, B or D genomes, a higher number of insertions in the B genome was detected compared to A or D genome, suggesting a specific amplification in the history of B genome progenitors. In conclusion, a novel LTR retrotransposon TriRe‒1 should be valuable for the development of molecular markers based on insertion polymorphism among the cultivars, and also the genome-specific TriRe‒1 insertion site can be utilized to study evolutional history of wheat genomes.　レトロトランスポゾンは植物ゲノムの主要な構成要素であり，コムギゲノムにおいてはその80ｵを占める．特に LTR 型レトロトランスポゾンの割合が高く，ゲノムの拡大，配列の多様性およびゲノム構造変異等に大きく寄与し てきたと考えられている．これら配列は自身のコピー配列を複製し増幅するため，ゲノム中には数百，数千に及ぶコ ピー配列をもつ．また，ゲノム進化の過程において多数のファミリーを形成してきた．これら多数のファミリーのう ち，現在でも転移活性を示す活性型ファミリーは，品種間において高い挿入多型を示すことが知られている．このよ うな挿入多型は，連鎖解析および系統解析等各種遺伝解析に利用可能である. 本研究では，コムギにおける新規活性 型レトロトランスポゾンファミリー TriRe-1 の特徴を詳細に解析した．TriRe-1 は転移に必要なタンパク質をコー ドする内部配列をもち，また日本で育成されたコムギ近縁品種間においても高い挿入多型を示したため，現在でも転 移活性を有している，もしくはごく最近まで転移していた可能性が高いと考えられた．一方で，コムギ染色体欠損系 統（ナリソミックテトラソミック系統）を用い，TriRe-1 の挿入箇所を比較解析した．その結果，大部分の挿入箇所 は複数の同祖染色体に存在すると考えられたが，Ｂゲノムにおいて最も多くの特異的な挿入箇所が同定された．よっ て，Ｂゲノム祖先種において活発に増幅してきた可能性が示唆された．今回の結果により，新規活性型レトロトラン スポゾン TriRe-1 の品種間挿入多型を利用した DNA マーカー，また，各ゲノム（Ａ，Ｂ，Ｄゲノム）特異的な挿 入箇所を利用したゲノム識別性に優れた DNA マーカーの開発の可能性が期待される

Correction: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams.

[This corrects the article DOI: 10.1371/journal.pone.0171274.]

A microsensing system for the in vivo real-time detection of local drug kinetics

International audienceReal-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 μm in diameter. We show that, in the guinea pig cochlea, the system can measure-simultaneously and in real time-changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected

Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams

<div><p>Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; <i>uvrA</i>, <i>uvrB</i>, <i>uvrC</i>, <i>uvrD</i>, <i>uvrD</i> paralog [<i>uvrD</i>p] and <i>mfd</i>) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While <i>uvrA</i>, <i>uvrD</i>p and <i>mfd</i> were conserved in all symbionts, <i>uvrB</i> and <i>uvrC</i> were degraded in all clade I symbionts but were apparently intact in clade II symbionts. <i>UvrD</i> was disrupted in two clade II symbionts. Among the intact genes in <i>Ca</i>. Vesicomyosocius okutanii (clade I), expressions of <i>uvrD</i> and <i>mfd</i> were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of <i>uvrA</i> and <i>uvrDp</i> were not. In contrast, all intact genes were expressed in the symbiont of <i>Calyptogena pacifica</i> (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from <i>Bathymodiolus septemdierum</i> were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and <i>mutY</i> in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome.</p></div

Deletion profiles of <i>uvrD</i> in the PCR products of vesicomyid clam symbionts.

<p>Vertical red lines on Cpac_S and Ifos_S indicate PCR primers redesigned for the second PCR. Gene arrangement and the direction of <i>uvrD</i> and neighboring genes of <i>Ca</i>. Ruthia magnifica are shown at the top, including Rmag_0319, hypothetical protein gene; Rmag_0320, <i>uvrD</i>; Rmag_0321, hypothetical protein gene; Rmag_3022, Lysine 2, 3-aminomutase YodO family protein gene. Bidirectional arrows in columns indicate the consensus helicase motifs. Symbols and demarcations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171274#pone.0171274.g001" target="_blank">Fig 1</a>.</p

Deletion profiles of <i>uvrB</i> in the PCR products of vesicomyid clam symbionts.

<p>ORFs of Rma are shown at the top: Rmag_0425, aminotransferase gene; Rmag_0427, Radical SAM domain protein. Red bars in the columns indicate stop codons. Horizontal dashed lines indicate the lost portion of the fragmented gene. The additional vertical red line on the right side of the column of Cfau_S indicates the position of the redesigned PCR primer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171274#pone.0171274.s002" target="_blank">S2 Table</a>). Lengths for PCR products are shown on the right side of each column. ORFs of <i>uvrB</i> in clade I symbionts are highly fragmented. Bidirectional arrows in columns indicate the consensus helicase motifs. Color gradient of the bidirectional arrows indicate the identity to that of <i>E</i>. <i>coli</i>. Symbionts are abbreviated as follows: Akaw_S, <i>A</i>. <i>kawamurai</i> symbiont; Clau_S, <i>C</i>. <i>laubieri</i> symbiont; Pkil_S, <i>P</i>. <i>kilmeri</i> symbiont; Psoy_S, <i>P</i>. <i>soyoae</i> symbiont; Vok, <i>Ca</i>. Vesicomyosocius okutanii (<i>P</i>. <i>okutanii</i> symbiont); Cpac_S, <i>C</i>. <i>pacifica</i> symbiont; Cfau_S, <i>C</i>. <i>fausta</i> symbiont; Cnau_S, <i>C</i>. <i>nautilei</i> symbiont; Pste_S, <i>P</i>. <i>stearnsii</i> symbiont; Rma, <i>Ca</i>. Ruthia magnifica (<i>C</i>. <i>magnifica</i> symbiont); Ifos_S, <i>I</i>. <i>fossajaponicum</i> symbiont; Apha_S, <i>A</i>. <i>phaseoliformis</i> symbiont; Bsep_S, <i>Bathymodiolus septemdierum</i> symbiont.</p

Probability matrix of round-robin paired t-test of GC content of all of the examined genes (lower left) and of 6 intact genes (upper right) in 12 vesicomyid clam symbionts.

<p>Probability matrix of round-robin paired t-test of GC content of all of the examined genes (lower left) and of 6 intact genes (upper right) in 12 vesicomyid clam symbionts.</p

Electrophorograms of the RT-PCR products of NER genes in <i>Ca</i>. Vesicomyosocius okutanii (Vok) and the symbiont of <i>C</i>. <i>pacifica</i> (Cpac_S).

<p>A, Cpac_S; B, Vok. Expression of NER-related genes was analyzed by RT-PCR. Lane 1, <i>uvrA</i>; lane 2, <i>uvrB</i> or corresponding DNA region; lane 3, <i>uvrC</i> or corresponding DNA region; lane 4, <i>uvrD</i>; lane 5, <i>uvrDp</i>; lane 6, <i>recA</i>; lane 7, <i>mfd</i>; lane 8, 16S rRNA gene; lane 9, 16S rRNA gene negative control (with RNase treatment before RT PCR). M, molecular markers. Primers and the predicted lengths of amplicons are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171274#pone.0171274.s003" target="_blank">S3 Table</a>. Red arrowheads indicate the position of a band where no signal was detected.</p

Deletion profiles of <i>uvrC</i> in vesicomyid clam symbionts.

<p>Open reading frames (ORFs) of <i>uvrC</i> are largely deleted in clade I symbionts. Lengths of the <i>uvrC</i> ORF and of the translated product are shown in the columns. The arrangement and direction of <i>uvrC</i> and neighboring genes of <i>Ca</i>. Ruthia magnifica are shown at the top: Rmag_0599, toluene tolerance protein; Rmag_0598, uvrC; Rmag_0597, hypothetical protein. Bidirectional arrows in columns indicate the consensus <i>uvrC</i> motifs. Other symbols and demarcations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171274#pone.0171274.g001" target="_blank">Fig 1</a>.</p