Abstract

Genome rearrangements such as indels, recombinations and inversions are one of the most important resources of adaptive evolution of life. These rearrangements can be induced by double strand breaks and the change in DNA modification status, which is the result of the activity of restriction-modification (RM) systems. To reveal the relationship between these two factors, genome rearrangements and RM systems, I adopted the genome comparison analysis, especially for the comparison of the Helicobacter pylori genome. This species are famous for variable and plastic genomes due to high rate of mutation and recombination, and also famous for possessing many RM systems. First, I classified genome inversions in 10 H. pylori genomes into four mechanisms, which include the novel mechanism of genome rearrangement called DNA duplication associated with inversion (DDAI). Second, RM systems in all prokaryote genome sequences are investigated and revealed the mobility of them through flanking repeat sequences in various configuration. Third, RM systems in H. pylori are systematically investigated and revealed the mobility of target recognition domains of RM genes in addition to the mobility of RM system itself. By these results, I revealed the generality of RM systems' mobility and their frequent involvement in genome rearrangements, which sometimes result in change of gene composition in genomes.

Results

Birth and death of genes linked to chromosomal inversion

Birth and death of genes by mechanism such as gene duplication are central to evolution, and yet the underlying mechanisms remain elusive. Availability of closely-related complete genome sequences now helps to trace gene copy number in relation to genome organization. The Gram-negative human stomach pathogen Helicobacter pylori is known for its plastic genome and geographical differentiation. We sequenced 4 Japanese H. pylori strains and analyzed with other 6 H. pylori genome sequences which were already available on database. Whole genome sequence comparison of H. pylori revealed a copy number change specific to East Asian strains in genes of outer membrane protein family. When the position of these genes are compared with the genome context, some of them were positioned at the endpoint of large genome inversions. I named this novel mechanism of genome rearrangements as DNA Duplication Associated with Inversion (DDAI, Figure 1). All the long genome inversions in 10 H. pylori genomes were analyzed and their mechanism were classified into 4 types: (i) DDAI, (ii) homologous recombination at long inverted repeat, (iii) recombination at short inverted repeat, and (iv) inversion adjacent to a mobile element. These analysis of long inversion events allowed reconstruction of synteny evolution in this species. These results may serve as a paradigm in analyzing long and short-term genome evolution in various organisms and in cancer cells thorough extensive DNA sequencing.

Putative mobile forms of restriction-modification systems and related rearrangements

The mobility of restriction-modification (RM) gene complexes and their association with genome rearrangements is a subject of active investigation. Here I conducted systematic genome comparisons and genome context analysis on fully sequenced prokaryotic genomes to detect RM-linked genome rearrangements. RM genes were frequently found to be linked to mobility-related genes such as integrase and transposase homologs. They were flanked by direct and inverted repeats at significantly high frequency (Figure 2). Insertion by long target duplication was observed for I, II, III, and IV restriction types. I found several RM genes flanked by long inverted repeats, some of which had apparently inserted into a genome with a short target duplication. In some cases, only a portion of an apparently complete RM system was flanked by inverted repeats (Figure 3).

Domain movement within a gene

A function of a protein is carried out by a specific domain localized in its specific position. In the present work, I report that a specific amino acid sequence can move between a domain at one position and a domain at another position within a same gene. This discovery was made during sequence comparison of restriction enzyme genes within a bacterial species called Helicobacter pylori. In the specificity subunit of Type I restriction enzymes, DNA sequence recognition is mediated by Target Recognition Domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location): these sequences appear to have moved between these two domains (Figure 4). The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y stand for the repeat sequences. The movement is likely realized by recombination at these flanking DNA repeats. In accord with this hypothesis, recombination at these repeats also appears to have decreased the two TRDs into one TRD, or increased into three (TRD1-TRD2-TRD2) and to have allowed TRD sequence movement between genes at different loci. Similar movements of a sequence between TRD1 and TRD2 were observed for the specificity subunit of a Type IIG restriction enzyme. The lateral domain movements within a protein, designated here as DOMO (domain movement), represent novel routes for diversification of proteins.

Figure 1 DDAI mechanism. (a) A genomic region represented as a wavy blue arrow is duplicated in inverted orientation at the opposite end of a chromosomal inversion. This is followed by inversion through homologous recombination involving the duplicated regions. (b) Nucleotide sequence alignments around the inversion break points. Only one of the two sequence alignment sets shows an overlap after DDAI. (c) Hypothetical mechanism of DDAI. DNA breakages are inserted at four positions, leading to generation of nick and double strand break. Strand exchange occurs with inversion, followed by closure of gaps by replication.

Figure 2 Frequency of genes flanked by (a) direct or inverted repeats, (b) direct repeats, and (c) inverted repeats. The vertical axis indicates% of the 11,554 compared RM-system-flanking sequence pairs. Black and white bars represent frequencies of flanking repeats and control genes, respectively. White circles indicate the ratio of RM systems to control genes for repeat frequency.

Figure 3 Transposon-like structure of RM systems flanked by repeat sequences. Triangles and arrows represent different sets of repeat sequences. (a) Type II RM genes in X. oryzae pv. oryzae KAC10331 are flanked by 65-bp inverted repeats (aligned below). The resulting unit is further flanked by 8-bp direct repeats (underlined), which are identical to the 8-bp sequence at the empty locus in X. oryzae pv. oryzae PX099A. The short direct repeat sequences are flanked by part of the predicted recognition sequence of the RM system (boxed) in the other genome. (b) Type II genes in N. gonorrhoeae NCCP 11945 are flanked by 26-bp inverted repeats (aligned below). The resulting unit is further flanked by 8-bp direct repeats, which are identical to the 8-bp sequence at the empty locus in N. meningitides MC58.

Figure 4 Diversity at target recognition domains of Group 2 specificity subunit. (A) Gene context of Group 2 specificity subunit. (B, C) Mechanisms of TRD exchange by homologous recombination. (D)Mechanism of TRD loss by homologous recombination. (E) Alleles of Group 2 specificity subunit paralogs at locus 1 and 2.

本論文は、ゲノム(遺伝情報)配列の比較による、遺伝子とゲノムの再編の新しい機構の発見について述べている。

Chapter 2では、「ゲノム内の逆位(反転)に伴って、その両端の領域がコピーされる」という、遺伝子重複の新しい機構を、同じ種に属する複数のゲノム配列の比較によって発見した。

図式化すると、

ー ある遺伝子 >>>>>>>>> ー

というゲノム内の配列から、

ーある遺伝子 <<<<<<<<< その遺伝子のコピー ー

が生じる。ここで、不等号の向きの反転は、ゲノムの一部の逆位を示している。

この機構により、遺伝子の誕生だけでなく、崩壊をも説明できた。この反応が重要であるのは、ゲノム中の遺伝子の増減が、遺伝子の機能の変化を通じて、適応進化において重要な役割を果たすためである。

対象の生物は、半数の人間の胃に住み着き、胃潰瘍、胃がんを起こすピロリ菌である。多数のゲノムの比較によって、逆位がこれを含む4つの機構で説明されることを示し、ゲノムの種内進化史の再構築に成功した。

この発見と解析法は、直ちにゲノム進化とがんゲノムの研究に役立つだけでなく、ゲノム科学、遺伝学、分子生物学、進化生物学とそれらの関連する医、バイオテクノロジー、環境などの諸分野に、インパクトを持つと予想される。実際、PNAS誌(インパクトファクター9.4)(2011年1月6日)に刊行の内容は、ただちにNature誌に取り上げられている(2011年1月13日)。

Chapter 3は、制限修飾系の「動き」について解析したものである。制限修飾系はDNA切断活性を持つ制限酵素の遺伝子と、DNAメチル化活性を持つ修飾酵素の遺伝子が隣り合って存在している遺伝子塊である。DNAのメチル化は、ゲノム情報の発現等エピジェネティクス(ATGC以外のDNAに伴う遺伝情報)に影響する。切断活性は、このメチル化というIDの無い外敵等のDNAを破壊する。制限酵素は生命科学の必須の道具でもある。本研究は制限修飾系の動きについて、データベース中にある全配列を用いて網羅的定量的に解析した。

制限修飾系が繰り返し配列に高頻度で挟まれることを証明した。また、制限修飾系の挿入機構の一般性を示した。さらに、トランスポゾン様の構造の制限修飾系を発見した。これらは、「制限修飾系が「動く遺伝子」である」という仮説を決定的に示す結果として重要である。論文はNucleic Acids Research(インパクトファクター7.5)に掲載された。

Chapter 4では、「一つの遺伝子内の複数のサイトの間で配列情報が動く」例を、種内(ピロリ菌)でのゲノム比較を通じて発見した。

ひとつのタンパク質遺伝子に次の二つの状態があった。

サイト1 サイト2

ーX配列 ーY配列ー

ーZ配列 ー X配列ー

さらに、配列のサイト間移動は、両方のサイトがともに両側にもっている配列a, bでの組換えによることが示唆された。

ーa サイト1 bーa サイト2 b ー

この遺伝子は、制限修飾系のためにDNA配列を認識するタンパク質を作っており、サイト1とサイト2は、それぞれDNA配列の左半分と右半分を読み取る。発見された配列移動によって、この制限修飾系が様々なDNA配列を認識できるようになり、制限酵素によるDNA切断活性と、修飾酵素のメチル化活性が多様化すること、それが、侵入DNAへの感染防御とエピジェネティクス状態の多様化に寄与することが推測できる。

本発見は、分子生物学において大きなインパクトを持つ。論文は現在投稿中である。

いずれの解析も、遺伝子とゲノムについての根本的に重要な発見を含んでおり、生命科学諸分野に大きく寄与する。

なお、本論文のChapter 2は阿部健太郎、小林一三、Chapter 3は河合幹彦、矢原耕史、高橋規子、半田直史、鶴剛史、大島健志朗、吉田優、東健、服部正平、内山郁夫、小林一三、Chapter 4は河合幹彦、内山郁夫、小林一三との共同研究であるが、論文提出者が主体となって分析および検証を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(生命科学)の学位を授与できると認める。