家蚕微孢子虫（Nosema bombycis）基因组学研究
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摘要
生命之所以不同于非生命物质,其中一个重要特征就在于它具有包括遗传和变异在内的进化。而作为分子进化和宏观进化的一个重要组成部分——水平基因转移(Horizontal Gene Transfer, HGT)——是指在近缘的、远缘的、甚至无亲缘关系的生物个体之间、细胞之间、或细胞内部的各细胞器之间,以及基因组内部所进行的遗传物质转移现象。这种现象广泛存在于自然界中,对物种基因组及其进化都起着十分重要的作用。目前,水平基因转移的研究主要集中在原核生物,而在真核生物基因组中水平基因转移的鉴定和研究尚处于滞后状态,仅在少数一些单细胞真核生物中取得了进展,而且它们的鉴定结果也都十分保守。其实真核生物同原核生物一样,也存在着大规模的水平基因转移事件,只是由于缺乏真核生物全基因组的系统检索方法而影响了其研究。有报道认为真菌是研究真核生物基因组中水平基因转移的一个优良谱系。
作为系统分类定位于真菌界的微孢子虫(Microsporidia),是一类专性寄生于细胞内的单细胞真核生物。它的宿主范围非常广,包括几乎所有的无脊椎动物和脊椎动物,甚至于人类自身,并能引发目前尚无药物根治的微孢子虫病,因此受到广泛关注。家蚕微孢子虫(Nosema bombycis)是最先被报道的、研究较为深入的一个典型种。近年,本实验室启动了家蚕微孢子虫的基因组测序计划,目前已经得到基因组7.8倍覆盖度的全基因组鸟枪法测序序列(whole genome shotgun, WGS、25倍覆盖度的Solexa测序数据、0.3倍覆盖度的miniBAC文库和含11,155条表达序列标签(expressed sequence tags, EST)的cDNA文库。预测家蚕微孢子虫基因组大小为15.7 Mb,18条染色体,包含4,305个编码基因。基于上述数据,开展了家蚕微孢子虫基因组中水平基因转移的研究,以期为在真核生物基因组中行之有效地鉴定水平基因转移提供一定参考,并为深入理解微孢子虫的基因组特征及其遗传进化模式,进而为预防和治疗微孢子虫病打下一定科学基础。
1.家蚕微孢子虫基因组中水平转移基因鉴定
基于家蚕微孢子虫全基因组数据,利用系统进化分析法和基于基因同源性全基因组统计的Darkhorse法,一共鉴定到55个可能水平转移基因。其中的48个(87.27%)被两种方法所共同确定,支持了该结果的可靠性。从这55个基因中随机抽取4个进行Southern杂交实验,结果表明这些水平转移基因确实存在于家蚕微孢子虫的基因组上,而非外来污染序列。
此外,Darkhorse法的结果还进一步证实了微孢子虫与真菌的亲缘关系。Southern杂交结果展现出预测拷贝数两倍的条带信号,推测可能是由于家蚕微孢子虫双核多态性原因造成的。
2.家蚕微孢子虫水平转移基因的基因特征及共线性
通过核苷酸组成、密码子偏好性、转录活性、选择压力等基因特征方面的分析,发现家蚕微孢子虫水平转移基因与其全基因组具有明显差异。例如,就全基因组的平均值而言,水平转移基因的GC含量更高、密码子偏好性更弱、最优密码子选择相悖且密码子GC含量也更高、转录水平偏低(大多没有EST支持,或支持条数少)、Ka／Ks值偏大(正选择和中性选择的情况偏多)等等。这些结果在一定程度上支持55个水平转移基因确实是外源的。
基于其他微孢子虫中同源基因的有无,将55个水平转移基因分为微孢子虫门物种分化前(the HGT before the divergence of microsporidia phylum, bHGT)和分化后(the HGT after the divergence of microsporidia phylum, aHGT)两类。而这一分类得到了上述基因特征、共线性分布图以及SOC (Self-Organizing Clustering)自主分类的支持,表明bHGT和aHGT确实存在差异。而且,相对于aHGT基因而言,bHGT更趋近于家蚕微孢子虫基因组。推测这可能是由于bHGT是在微孢子虫门物种分化前就已经转移进入了微孢子虫共同祖先的基因组中,随着物种进化,其原供体的基因组特征被逐渐消减,并慢慢地与微孢子虫基因组发生协同进化,逐渐趋向于微孢子虫的基因组特征,这正是水平转移基因的“同质化”(amelioration)过程；相对而言,aHGT进入家蚕微孢子虫基因组的时间较短,“同质化”效果尚不明显。
3.家蚕微孢子虫水平转移基因的来源物种及转移时间
家蚕微孢子虫水平转移基因的来源谱系很广,基本上大多数物种门都有来源,认为家蚕微孢子虫水平基因转移的来源物种可能是无差别的。其中,占比例最大的是细菌域(83.64%),这与目前大多数真核生物基因组报道的水平基因转移情况一致,推测是由于细菌类群大、种类多、生活环境多种多样、数据库中序列信息较多等原因造成的。
检测到的水平转移方向都是外源物种向微孢子虫或家蚕微孢子虫转移,却没发现反过来的情况。认为可能是由于真核生物转移机制少、基因组大、且基因比较复杂等原因造成的。
定量分析表明,12个bHGT的转移时间范围位于357～760百万年间,而3个aHGT的在11～73百万年间。由于上述数据量少,进而根据核苷酸组成差异进行定性分析,得出bHGT的转移时间大约是aHGT基因的1.55倍。肯定了bHGT的进化时间确实比aHGT的久远,再次支持了它们的分类。
4.家蚕微孢子虫水平转移基因的功能作用
大多数家蚕微孢子虫水平转移基因的功能未知,但在己知部分,代谢功能相关基因所占比例最大。这种情况同于己报道的溶组织内阿米巴(Entamoeba histolytica)和阴道毛滴虫(Trichomonas vaginalis).认为这是由于水平基因转移具有简单性造成的,即相对于信息基因而言,操纵基因(如代谢功能基因)更为简单,更利于水平转移基因的获得。
aHGT在基因组中主要扮演功能新增角色,而bHGT起功能补充作用。推测水平转移基因进入基因组,为该物种提供新基因新功能做出了巨大贡献,随着物种进化,外源基因需要适应基因组或在正选择压力下才能保留下来。因此,提供新功能的水平转移基因比扮演补充角色的更有被选择筛除掉的危险。故而,在物种分化之前就进入基因组的bHGT,在长期的选择作用下,保留下来更多的是只起到补充功能作用的基因。水平转移基因进入基因组事件与不适应基因组的基因被选择事件是同时发生着的,现今基因组中的水平基因转移是两事件动态平衡后的结果。
此外,在甲羟戊酸途径中,发现一个水平转移基因起到了联通整条通路的作用。表明水平转移基因对物种的生命活动,尤其是代谢功能方面,发挥着重要作用。
5.家蚕微孢子虫单个水平转移基因的具体分析
通过对家蚕微孢子虫锰超氧化物歧化酶基因(NbMnSOD)进行分析,发现SOD基因的系统进化树、蛋白质结构和亚细胞定位之间存在着一定联系,且三者的结果都表明NbMnSoD来源于某一变形菌门(proteobacteria)物种的水平基因转移。
NbMnSOD发生了串联重复事件,且重复基因比原基因序列变异更大、更不保守、进化更快,结构特征也有很大变化。意味着该基因可能已经进化出新功能,而且选择压力结果推测该进化是由自然选择所控制的。
虽然丝氨酸蛋白酶抑制剂基因(serpin)分布广泛,但都只是在少数一些物种中零星存在,认为这种不连续分布也只有通过水平基因转移现象才能得以解释。结合系统进化分析,推测家蚕微孢子虫serpin可能来源于一种痘病毒(poxViridae)物种。
6.家蚕微孢子虫基因组中转座元件分析
利用多种软件和数据库,鉴定了基因组内的水平基因转移——转座元件。家蚕微孢子虫基因组中的转座元件占据了相当大的比例(36.12%),认为是导致其基因组偏大的原因之一
大部分转座元件均属于未报道的新类型,而已知中的最大一类是Ty3／Gypsy型LTR反转座元件。基于以前研究,新的6个具转录活性的家蚕微孢子虫LTR反转座元件(Nbr)被鉴定,表明它们仍然发生着转座并起着改造基因组的作用。另外,转座元件的拷贝数少、多态性高、结构域不完整等特点,又说明缺失和丢失事件也发生在这些转座元件中。这种矛盾现象己被报道,认为是基因组中转座元件进行转座和发生消失两种事件的动态平衡结果,并且这一平衡受到自然选择的调控。
通常,为了最大程度降低转座事件对基因组造成的破坏,转座元件受到的多是强烈负选择压力,而且它们的转座位点多位于两不相关的共线性区域之间,对基因组确实影响不大。然而,Nbrll却不同于常规转座元件,它整合进了一个保守共线性区域内部,且可能导致了基因组重排。进一步分析认为该变异可能适应了基因组,并对物种有利,因此Nbrll在正选择压力下保留了下来。
相对其他微孢子虫而言,家蚕微孢子虫可能由于寄生宿主不同等原因,基因组中可以存在着大量转座元件,且它们有活性有作用。甚至,具有一些来自于其宿主家蚕的转座元件,就更加说明转座元件在基因组及其进化上起着重要作用。相对于其他己报道水平基因转移的物种,家蚕微孢子虫中鉴定到的水平转移基因所占基因组比例更大,其中一个重要原因可能就在于转座元件使得水平转移基因能更容易的插入并整合到基因组中、以及促进了水平转移基因在基因组中的传播。而这一推测也得到了利用二者距离和多基因组统计所得出的基因组中水平基因转移与转座元件具有正相关性结论的支持。
Horizontal gene transfer (HGT), which is defined as the movement of genetic material between different organisms, cells, organelles and even DNA within the genome, plays a highly significant role in the evolution of both the species and their genome. To date, a great number of the studies on HGT in the prokaryotic genomes are finished, but the ones in the eukaryotic genomes fall behind because the methods of detecting HGT in them are hard, and just several unicellular eukaryotes identified the horizontally transferred genes. There repersents in a report claimed that fungi will be a compatible module to inquiry the eukaryotic HGT.
Related to the fungi kingdom, microsporidia are a group of obligate intracellular eukaryotic parasites that infect a wide variety of species, including humans. The microsporidiosis caused by them are severe and hard to cure radically, thus microsporidia draws a great interest to the scientists around the world. Nosema bombycis, as the first discovered microsporidia, have been studied since the middle of the nineteenth century. In 2003, the genome project of Nosema bombycis was started in our laboratory. So far, a whole genome shotgun database with 7.8-fold coverage of the haploid genome, a Solexa database with 25-fold coverage, a miniBAC library with 0.3-fold coverage and a cDNA library with 11,155 expressed sequence tags were constructed successfully. Based on these databases, we carried out the analyses of horizontal gene transfer within the genome of N. bombycis.
1. Identification of horizontally transferred genes in the N. bombycis genome
Based on the whole genome database of N. bombycis, we identified 55 horizontally transferred genes using the phylogenetic analysis and Darkhorse method. Forty-eight of them were confirmed by both methods, indicative of a reliable result. We randomly chose four of them to carry out the Southern blot, and the result shows that these genes exist in the genome of N. bombycis rather than the contamination.
2. Characterization of horizontally transferred genes in the N. bombycis genome
There are lots of differences between the features of horizontally transferred genes and the ones of whole genomic genes, such as nucleotide composition, codon usage bias, transcriptional activity and selection pressure. For example, the horizontally transferred genes show higher GC content, weaker codon usage bias, different optimal codon, usage and their higher GC content, lower transcription and bigger Ka/Ks value than the whole genomic genes. These results reconfirm that the N. bombycis horizontally transferred genes are exogenous.
According to the status of the homologues of horizontally transferred genes in the other microsporidian genomes, we divided 55 these genes into two categories:ones before the divergence of microsporidia phylum (bHGT); while the other is after that divergence (aHGT). Moreover, this classification was supported by the above-mentioned features, the syntenic maps and the self-organizing clustering method. Compared with aHGT genes, the features of bHGT are more similar to the ones of whole genomic genes, suggesting that there is more time for bHGT genes to change themselves to adapt to their host genome, and this is the so-called amelioration process of horizontally transferred genes.
3. Source species, transferred direction and transferred time of horizontally transferred genes in the N. bombycis genome
Taxonomic distribution of 55 horizontally transferred genes was identified. All of them are prokaryote/virus-to-eukaryote, but no genes origined from its host Bombyx mori were found. The majority of such entries have one or more members of the proteobacteria as the most possible origin in aHGT while firmicutes in bHGT. This distribution of N. bombycis horizontally transferred genes is widespread, where transfer events from many phylums of both Bacteria and Archaea domains of life plus Viruses are uncovered, in a highly resolved phylogenetic tree of life.
All of the detected transferred directions are the HGT from other organism to microsporidia or N. bombycis. The calculated transferred time of bHGT show 357-760 MYr (million years) while the one of aHGT show 11-73 MYr. Further qualitative analysis show that the transferred time of bHGT is 1.55-fold than the one of aHGT, fully affirming the classification of bHGT and aHGT.
4. Function of horizontally transferred genes in the N. bombycis genome
Most horizontally transferred genes of N. bombycis are functionally unknown, while the definitive one play role in many functions, specifically in metabolism. This phenomenon is similar with the known HGT from the genomes of Entamoeba histolytica and Trichomonas vaginalis, which can be interpreted by the hypothesis that HGT favors the acquisition of operational genes but not informational genes.
Generally speaking, bHGT genes play the supplementary role in the genome of N. bombycis, while aHGT take the newly-increased function. In addition, among the KEGG metabolic maps of 55 horizontally transferred genes, we discovered that the phosphomevalonate kinase (EC 2.7.4.2) encoded by a horizontally transferred gene play an important role in the mevalonate pathway.
5. Two cases of horizontally transferred genes in the N. bombycis genome
A comprehensive analysis of manganese superoxide dismutase (MnSOD) sequences from eukaryotes, Archaea and bacteria shows the microsporidial MnSODs to be grouped within bacteria MnSODs rather than eukarotic MnSODs, in terms of sequence similarity and predicted oligomeric structure and subcellular localization, indicative of a possible horizontal gene transfer.
Tandem duplicated NbMnSOD2 gene seems to have changed more rapidly and is under less evolutionary constraint than the original NbMnSODl. NbMnSOD2 shows some sequence properties of tetramers and other characteristics quite different from NbMnSODl. This suggests that NbMnSOD2 may function under different conditions or in different tissues of its host rather than simply resulting in an increase in expression.
The serine proteinase inhibitors (serpin) genes were found in a few organisms of many wide phylums, inferring that only the HGT can be used to explain this circumstance. Thus, we suggested that the serpin of N. bombycis had possibly come from a poxviridae according to its phylogenetic analysis.
6. Transposable elements in the N. bombycis genome
As the horizontal gene transfer within the genome, transposable elements took account of 36.12%of whole genome sequences in the N. bombycis genome, suggesting this is one of the reasons for the bigger genome size of N. bombycis than other microsporidia. We also found four transposable elements were acquired from its host silkworm by horizontal transfer. The considerable transposable elements are an important factor for the reshuffling and evolution of the N. bombycis genome. Moreover, a positive correlation between the transposable elements and horizontally transferred genes was observed by our statistical analysis.
The bulk of transposable elements are unclassed, and the Ty3/gypsy LTR retrotransposons (Nbr), which constitute the largest part of known classes, have been studied already. Following previously reported eight Nbr elements, we detected six novel transcribed ones (Nbr9-Nbr14). Retrotransposon diversity and incomplete domains with insertions, deletions and in-frame stop codons in coding regions were detected in them, suggesting that both defective and loss events of LTR retrotransposon have happened in N. bombycis genome. Analysis of selection showed that strong purifying selection acts on all elements except Nbrl 1. This implies that selective pressure keeps both these Nbrs and their functions in genome. Interestingly, Nbrl 1 is under positive selection and some positively selected codons were identified, indicating that new functionality might have evolved in the Nbrl 1 retrotransposon. Unlike other transposable elements, Nbrl 1 has integrated into a conserved syntenic block and probably resulted in the inversion of both flanking regions. This demonstrates that transposable elements are an important factor for the reshuffling and evolution of their host genomes, and may be maintained under natural selection.
作为系统分类定位于真菌界的微孢子虫(Microsporidia),是一类专性寄生于细胞内的单细胞真核生物。它的宿主范围非常广,包括几乎所有的无脊椎动物和脊椎动物,甚至于人类自身,并能引发目前尚无药物根治的微孢子虫病,因此受到广泛关注。家蚕微孢子虫(Nosema bombycis)是最先被报道的、研究较为深入的一个典型种。近年,本实验室启动了家蚕微孢子虫的基因组测序计划,目前已经得到基因组7.8倍覆盖度的全基因组鸟枪法测序序列(whole genome shotgun, WGS、25倍覆盖度的Solexa测序数据、0.3倍覆盖度的miniBAC文库和含11,155条表达序列标签(expressed sequence tags, EST)的cDNA文库。预测家蚕微孢子虫基因组大小为15.7 Mb,18条染色体,包含4,305个编码基因。基于上述数据,开展了家蚕微孢子虫基因组中水平基因转移的研究,以期为在真核生物基因组中行之有效地鉴定水平基因转移提供一定参考,并为深入理解微孢子虫的基因组特征及其遗传进化模式,进而为预防和治疗微孢子虫病打下一定科学基础。
1.家蚕微孢子虫基因组中水平转移基因鉴定
基于家蚕微孢子虫全基因组数据,利用系统进化分析法和基于基因同源性全基因组统计的Darkhorse法,一共鉴定到55个可能水平转移基因。其中的48个(87.27%)被两种方法所共同确定,支持了该结果的可靠性。从这55个基因中随机抽取4个进行Southern杂交实验,结果表明这些水平转移基因确实存在于家蚕微孢子虫的基因组上,而非外来污染序列。
此外,Darkhorse法的结果还进一步证实了微孢子虫与真菌的亲缘关系。Southern杂交结果展现出预测拷贝数两倍的条带信号,推测可能是由于家蚕微孢子虫双核多态性原因造成的。
2.家蚕微孢子虫水平转移基因的基因特征及共线性
通过核苷酸组成、密码子偏好性、转录活性、选择压力等基因特征方面的分析,发现家蚕微孢子虫水平转移基因与其全基因组具有明显差异。例如,就全基因组的平均值而言,水平转移基因的GC含量更高、密码子偏好性更弱、最优密码子选择相悖且密码子GC含量也更高、转录水平偏低(大多没有EST支持,或支持条数少)、Ka／Ks值偏大(正选择和中性选择的情况偏多)等等。这些结果在一定程度上支持55个水平转移基因确实是外源的。
基于其他微孢子虫中同源基因的有无,将55个水平转移基因分为微孢子虫门物种分化前(the HGT before the divergence of microsporidia phylum, bHGT)和分化后(the HGT after the divergence of microsporidia phylum, aHGT)两类。而这一分类得到了上述基因特征、共线性分布图以及SOC (Self-Organizing Clustering)自主分类的支持,表明bHGT和aHGT确实存在差异。而且,相对于aHGT基因而言,bHGT更趋近于家蚕微孢子虫基因组。推测这可能是由于bHGT是在微孢子虫门物种分化前就已经转移进入了微孢子虫共同祖先的基因组中,随着物种进化,其原供体的基因组特征被逐渐消减,并慢慢地与微孢子虫基因组发生协同进化,逐渐趋向于微孢子虫的基因组特征,这正是水平转移基因的“同质化”(amelioration)过程；相对而言,aHGT进入家蚕微孢子虫基因组的时间较短,“同质化”效果尚不明显。
3.家蚕微孢子虫水平转移基因的来源物种及转移时间
家蚕微孢子虫水平转移基因的来源谱系很广,基本上大多数物种门都有来源,认为家蚕微孢子虫水平基因转移的来源物种可能是无差别的。其中,占比例最大的是细菌域(83.64%),这与目前大多数真核生物基因组报道的水平基因转移情况一致,推测是由于细菌类群大、种类多、生活环境多种多样、数据库中序列信息较多等原因造成的。
检测到的水平转移方向都是外源物种向微孢子虫或家蚕微孢子虫转移,却没发现反过来的情况。认为可能是由于真核生物转移机制少、基因组大、且基因比较复杂等原因造成的。
定量分析表明,12个bHGT的转移时间范围位于357～760百万年间,而3个aHGT的在11～73百万年间。由于上述数据量少,进而根据核苷酸组成差异进行定性分析,得出bHGT的转移时间大约是aHGT基因的1.55倍。肯定了bHGT的进化时间确实比aHGT的久远,再次支持了它们的分类。
4.家蚕微孢子虫水平转移基因的功能作用
大多数家蚕微孢子虫水平转移基因的功能未知,但在己知部分,代谢功能相关基因所占比例最大。这种情况同于己报道的溶组织内阿米巴(Entamoeba histolytica)和阴道毛滴虫(Trichomonas vaginalis).认为这是由于水平基因转移具有简单性造成的,即相对于信息基因而言,操纵基因(如代谢功能基因)更为简单,更利于水平转移基因的获得。
aHGT在基因组中主要扮演功能新增角色,而bHGT起功能补充作用。推测水平转移基因进入基因组,为该物种提供新基因新功能做出了巨大贡献,随着物种进化,外源基因需要适应基因组或在正选择压力下才能保留下来。因此,提供新功能的水平转移基因比扮演补充角色的更有被选择筛除掉的危险。故而,在物种分化之前就进入基因组的bHGT,在长期的选择作用下,保留下来更多的是只起到补充功能作用的基因。水平转移基因进入基因组事件与不适应基因组的基因被选择事件是同时发生着的,现今基因组中的水平基因转移是两事件动态平衡后的结果。
此外,在甲羟戊酸途径中,发现一个水平转移基因起到了联通整条通路的作用。表明水平转移基因对物种的生命活动,尤其是代谢功能方面,发挥着重要作用。
5.家蚕微孢子虫单个水平转移基因的具体分析
通过对家蚕微孢子虫锰超氧化物歧化酶基因(NbMnSOD)进行分析,发现SOD基因的系统进化树、蛋白质结构和亚细胞定位之间存在着一定联系,且三者的结果都表明NbMnSoD来源于某一变形菌门(proteobacteria)物种的水平基因转移。
NbMnSOD发生了串联重复事件,且重复基因比原基因序列变异更大、更不保守、进化更快,结构特征也有很大变化。意味着该基因可能已经进化出新功能,而且选择压力结果推测该进化是由自然选择所控制的。
虽然丝氨酸蛋白酶抑制剂基因(serpin)分布广泛,但都只是在少数一些物种中零星存在,认为这种不连续分布也只有通过水平基因转移现象才能得以解释。结合系统进化分析,推测家蚕微孢子虫serpin可能来源于一种痘病毒(poxViridae)物种。
6.家蚕微孢子虫基因组中转座元件分析
利用多种软件和数据库,鉴定了基因组内的水平基因转移——转座元件。家蚕微孢子虫基因组中的转座元件占据了相当大的比例(36.12%),认为是导致其基因组偏大的原因之一
大部分转座元件均属于未报道的新类型,而已知中的最大一类是Ty3／Gypsy型LTR反转座元件。基于以前研究,新的6个具转录活性的家蚕微孢子虫LTR反转座元件(Nbr)被鉴定,表明它们仍然发生着转座并起着改造基因组的作用。另外,转座元件的拷贝数少、多态性高、结构域不完整等特点,又说明缺失和丢失事件也发生在这些转座元件中。这种矛盾现象己被报道,认为是基因组中转座元件进行转座和发生消失两种事件的动态平衡结果,并且这一平衡受到自然选择的调控。
通常,为了最大程度降低转座事件对基因组造成的破坏,转座元件受到的多是强烈负选择压力,而且它们的转座位点多位于两不相关的共线性区域之间,对基因组确实影响不大。然而,Nbrll却不同于常规转座元件,它整合进了一个保守共线性区域内部,且可能导致了基因组重排。进一步分析认为该变异可能适应了基因组,并对物种有利,因此Nbrll在正选择压力下保留了下来。
相对其他微孢子虫而言,家蚕微孢子虫可能由于寄生宿主不同等原因,基因组中可以存在着大量转座元件,且它们有活性有作用。甚至,具有一些来自于其宿主家蚕的转座元件,就更加说明转座元件在基因组及其进化上起着重要作用。相对于其他己报道水平基因转移的物种,家蚕微孢子虫中鉴定到的水平转移基因所占基因组比例更大,其中一个重要原因可能就在于转座元件使得水平转移基因能更容易的插入并整合到基因组中、以及促进了水平转移基因在基因组中的传播。而这一推测也得到了利用二者距离和多基因组统计所得出的基因组中水平基因转移与转座元件具有正相关性结论的支持。
Horizontal gene transfer (HGT), which is defined as the movement of genetic material between different organisms, cells, organelles and even DNA within the genome, plays a highly significant role in the evolution of both the species and their genome. To date, a great number of the studies on HGT in the prokaryotic genomes are finished, but the ones in the eukaryotic genomes fall behind because the methods of detecting HGT in them are hard, and just several unicellular eukaryotes identified the horizontally transferred genes. There repersents in a report claimed that fungi will be a compatible module to inquiry the eukaryotic HGT.
Related to the fungi kingdom, microsporidia are a group of obligate intracellular eukaryotic parasites that infect a wide variety of species, including humans. The microsporidiosis caused by them are severe and hard to cure radically, thus microsporidia draws a great interest to the scientists around the world. Nosema bombycis, as the first discovered microsporidia, have been studied since the middle of the nineteenth century. In 2003, the genome project of Nosema bombycis was started in our laboratory. So far, a whole genome shotgun database with 7.8-fold coverage of the haploid genome, a Solexa database with 25-fold coverage, a miniBAC library with 0.3-fold coverage and a cDNA library with 11,155 expressed sequence tags were constructed successfully. Based on these databases, we carried out the analyses of horizontal gene transfer within the genome of N. bombycis.
1. Identification of horizontally transferred genes in the N. bombycis genome
Based on the whole genome database of N. bombycis, we identified 55 horizontally transferred genes using the phylogenetic analysis and Darkhorse method. Forty-eight of them were confirmed by both methods, indicative of a reliable result. We randomly chose four of them to carry out the Southern blot, and the result shows that these genes exist in the genome of N. bombycis rather than the contamination.
2. Characterization of horizontally transferred genes in the N. bombycis genome
There are lots of differences between the features of horizontally transferred genes and the ones of whole genomic genes, such as nucleotide composition, codon usage bias, transcriptional activity and selection pressure. For example, the horizontally transferred genes show higher GC content, weaker codon usage bias, different optimal codon, usage and their higher GC content, lower transcription and bigger Ka/Ks value than the whole genomic genes. These results reconfirm that the N. bombycis horizontally transferred genes are exogenous.
According to the status of the homologues of horizontally transferred genes in the other microsporidian genomes, we divided 55 these genes into two categories:ones before the divergence of microsporidia phylum (bHGT); while the other is after that divergence (aHGT). Moreover, this classification was supported by the above-mentioned features, the syntenic maps and the self-organizing clustering method. Compared with aHGT genes, the features of bHGT are more similar to the ones of whole genomic genes, suggesting that there is more time for bHGT genes to change themselves to adapt to their host genome, and this is the so-called amelioration process of horizontally transferred genes.
3. Source species, transferred direction and transferred time of horizontally transferred genes in the N. bombycis genome
Taxonomic distribution of 55 horizontally transferred genes was identified. All of them are prokaryote/virus-to-eukaryote, but no genes origined from its host Bombyx mori were found. The majority of such entries have one or more members of the proteobacteria as the most possible origin in aHGT while firmicutes in bHGT. This distribution of N. bombycis horizontally transferred genes is widespread, where transfer events from many phylums of both Bacteria and Archaea domains of life plus Viruses are uncovered, in a highly resolved phylogenetic tree of life.
All of the detected transferred directions are the HGT from other organism to microsporidia or N. bombycis. The calculated transferred time of bHGT show 357-760 MYr (million years) while the one of aHGT show 11-73 MYr. Further qualitative analysis show that the transferred time of bHGT is 1.55-fold than the one of aHGT, fully affirming the classification of bHGT and aHGT.
4. Function of horizontally transferred genes in the N. bombycis genome
Most horizontally transferred genes of N. bombycis are functionally unknown, while the definitive one play role in many functions, specifically in metabolism. This phenomenon is similar with the known HGT from the genomes of Entamoeba histolytica and Trichomonas vaginalis, which can be interpreted by the hypothesis that HGT favors the acquisition of operational genes but not informational genes.
Generally speaking, bHGT genes play the supplementary role in the genome of N. bombycis, while aHGT take the newly-increased function. In addition, among the KEGG metabolic maps of 55 horizontally transferred genes, we discovered that the phosphomevalonate kinase (EC 2.7.4.2) encoded by a horizontally transferred gene play an important role in the mevalonate pathway.
5. Two cases of horizontally transferred genes in the N. bombycis genome
A comprehensive analysis of manganese superoxide dismutase (MnSOD) sequences from eukaryotes, Archaea and bacteria shows the microsporidial MnSODs to be grouped within bacteria MnSODs rather than eukarotic MnSODs, in terms of sequence similarity and predicted oligomeric structure and subcellular localization, indicative of a possible horizontal gene transfer.
Tandem duplicated NbMnSOD2 gene seems to have changed more rapidly and is under less evolutionary constraint than the original NbMnSODl. NbMnSOD2 shows some sequence properties of tetramers and other characteristics quite different from NbMnSODl. This suggests that NbMnSOD2 may function under different conditions or in different tissues of its host rather than simply resulting in an increase in expression.
The serine proteinase inhibitors (serpin) genes were found in a few organisms of many wide phylums, inferring that only the HGT can be used to explain this circumstance. Thus, we suggested that the serpin of N. bombycis had possibly come from a poxviridae according to its phylogenetic analysis.
6. Transposable elements in the N. bombycis genome
As the horizontal gene transfer within the genome, transposable elements took account of 36.12%of whole genome sequences in the N. bombycis genome, suggesting this is one of the reasons for the bigger genome size of N. bombycis than other microsporidia. We also found four transposable elements were acquired from its host silkworm by horizontal transfer. The considerable transposable elements are an important factor for the reshuffling and evolution of the N. bombycis genome. Moreover, a positive correlation between the transposable elements and horizontally transferred genes was observed by our statistical analysis.
The bulk of transposable elements are unclassed, and the Ty3/gypsy LTR retrotransposons (Nbr), which constitute the largest part of known classes, have been studied already. Following previously reported eight Nbr elements, we detected six novel transcribed ones (Nbr9-Nbr14). Retrotransposon diversity and incomplete domains with insertions, deletions and in-frame stop codons in coding regions were detected in them, suggesting that both defective and loss events of LTR retrotransposon have happened in N. bombycis genome. Analysis of selection showed that strong purifying selection acts on all elements except Nbrl 1. This implies that selective pressure keeps both these Nbrs and their functions in genome. Interestingly, Nbrl 1 is under positive selection and some positively selected codons were identified, indicating that new functionality might have evolved in the Nbrl 1 retrotransposon. Unlike other transposable elements, Nbrl 1 has integrated into a conserved syntenic block and probably resulted in the inversion of both flanking regions. This demonstrates that transposable elements are an important factor for the reshuffling and evolution of their host genomes, and may be maintained under natural selection.
引文
[I]Bansal A K,Meyer T E. Evolutionary analysis by whole-genome comparisons. J Bacteriol,2002,184 (8): 2260-2272.
[2]Ochman H, Lawrence J G,Groisman E A. Lateral gene transfer and the nature of bacterial innovation. Nature, 2000,405 (6784):299-304.
[3]Keeling P J,Palmer J D. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet,2008,9 (8):605-618.
[4]Woese C. The universal ancestor. Proc Natl Acad Sci USA,1998,95 (12):6854-6859.
[5]Doolittle W F. Phylogenetic classification and the universal tree. Science,1999,284 (5423):2124-2128.
[6]Smets B F,Barkay T. Horizontal gene transfer:perspectives at a crossroads of scientific disciplines. Nat Rev Microbiol,2005,3 (9):675-678.
[7]Choi I G,Kim S H. Global extent of horizontal gene transfer. Proc Natl Acad Sci USA,2007,104 (11): 4489-4494.
[8]Ricard G, McEwan N R, Dutilh B E, et al. Horizontal gene transfer from Bacteria to rumen Ciliates indicates adaptation to their anaerobic, carbohydrates-rich environment. BMC Genomics,2006,7 (1):22.
[9]Syvanen M. Recent emergence of the modern genetic code:a proposal. Trends Genet,2002,18 (5):245-248.
[10]Jain R, Rivera M C,Lake.J A. Horizontal gene transfer among genomes:the complexity hypothesis. Proc Natl Acad Sci USA,1999,96 (7):3801-3806.
[11]Eisen J A. Horizontal gene transfer among microbial genomes:new insights from complete genome analysis. Curr Opin Genet Dev,2000,10 (6):606-611.
[12]Lawrence J G,Ochman H. Amelioration of Bacterial Genomes:Rates of Change and Exchange. J Mol Biol, 1997,44 (4):383-397.
[13]Mosig G Recombination and recombination-dependent DNA replication in bacteriophage T4. Annu Rev Genet, 1998,32(1):379-413.
[14]Jain R, Rivera M C, Moore J E, et al. Horizontal gene transfer accelerates genome innovation and evolution. Mol Biol Evol,2003,20 (10):1598-1602.
[15]Gogarten J P,Townsend J P. Horizontal gene transfer, genome innovation and evolution. Nat Rev Microbiol, 2005,3 (9):679-687.
[16]Martin W. Mosaic bacterial chromosomes:a challenge en route to a tree of genomes. Bioessays,1999,21 (2): 99-104.
[17]Griffith F. The signi cance of pneumococcal types. J Hyg,1928,27 113-159.
[18]Avery O T, McLeod C M,McCarthy M. Studies on the Chemical nature of the substance inducing transformation of pneumococcal types:Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med,1944,79 (2):137-158.
[19]McClintock B. The origin and behavior of mutable loci in maize. Proc Natl Acad Sci USA,1950,36 (6): 344-355.
[20]Lederberg J,Tatum E L. Novel genotypes in mixed cultures of biochemical mutants of bacteria, cold spring harb symp quant biol,1946,11 113-114.
[21]Ochiai K, Yamanaka T, Kimura K, et al. Inheritance of drug resistance (and its transfer) between Shigella strains and between Shigella and E. coli strains. Nihon Iji Shimpo 1861,1959,34
[22]Shapiro J A. Mutations caused by the insertion of genetic material into the galactose operon of Escherichia coli. J Mol Biol,1969,40 (1):93-105.
[23]Went F W. Parallel evolution. Taxon,1971,20 (2):197-226.
[24]Hiraizumi Y. Spontaneous recombination in Drosophila melanogaster males. Proc Natl Acad Sci U S A,1971, 68 (2):268-270.
[25]Green M M. Genetic instability in Drosophila melanogaster. De novo induction of putative insertion mutations. Proc Natl Acad Sci USA,1977,74 (8):3490-3493.
[26]Syvanen M. Cross-species gene transfer; implications for a new theory of evolution. J Theor Biol,1985,112 (2): 333-343.
[27]Ho M W, Ryan A,Cummins J. Cauliflower Mosaic Viral Promoter-A Recipe for Disaster? Microbial Ecology in Health and Disease,1999,11 (4):194-197.
[28]Gogarten P. Horizontal Gene Transfer:A New Paradigm for Biology. Esalen Center for Theory and Research Conference,2000,
[29]Bergthorsson U, Adams K L, Thomason B, et al. Widespread horizontal transfer of mitochondrial genes in flowering plants. Nature,2003,424 (6945):197-201.
[30]Rivera M C,Lake J A. The ring of life provides evidence for a genome fusion origin of eukaryotes. Nature,2004, 431 (7005):152-155.
[31]Bapteste E, Susko E, Leigh J, et al. Do orthologous gene phylogenies really support tree-thinking? BMC Evol Biol,2005,5(1):33.
[32]Huang J, Mullapudi N, Lancto C, et al. Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum. Genome Biol,2004,5 (11):R88.
[33]Loftus B, Anderson I, Davies R, et al. The genome of the protist parasite Entamoeba histolytica. Nature,2005, 433 (7028):865-868.
[34]Eichinger L, Pachebat J A, Glockner G, et al. The genome of the social amoeba Dictyostelium discoideum. Nature,2005,435 (7038):43-57.
[35]Berriman M, Ghedin E, Hertz-Fowler C, et al. The genome of the African trypanosome Trypanosoma brucei. Science,2005,309 (5733):416-422.
[36]Derelle E, Ferraz C, Rombauts S, et al. Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc Natl Acad Sci U S A,2006,103 (31):11647-11652.
[37]Carlton J M, Hirt R P, Silva J C, et al. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science,2007,315 (5809):207-212.
[38]Gladyshev E A, Meselson M,Arkhipova I R. Massive horizontal gene transfer in bdelloid rotifers. Science,2008, 320(5880):1210-1213.
[39]Winner M. Historic perspective on the microsporidia:expanding horizons. In:The microsporidia and microsporidiosis. Amer Society for Microbiology,1999.
[40]Larsson J I R. Identification of microsporidia. Acta protozoologica,1999,38 (3):161-197.
[41]Weber R, Bryan R T, Schwartz D A, et al. Human microsporidial infections. Clin Microbiol Rev,1994,7 (4): 426-461.
[42]Mathis A, Weber R,Deplazes P. Zoonotic potential of the microsporidia. Clin Microbiol Rev,2005,18 (3): 423-445.
[43]Visvesvara G S, Belloso M, Moura H, et al. Isolation of Nosema algerae from the cornea of an immunocompetent patient. J Eukaryot Microbiol,1999,46 (5):10S.
[44]Svenungsson B, Capraru T, Eveng rd B, et al. Intestinal microsporidiosis in a HIV-seronegative patient. Scand J Infect Dis,1998,30 (3):314-316.
[45]Lopez-Velez R, Turrientes M C, Garron C, et al. Microsporidiosis in travelers with diarrhea from the tropics. J Travel Med,1999,6 (4):223-227.
[46]Katinka M D, Duprat S, Cornillot E, et al. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature,2001,414 (6862):450-453.
[47]Sprague V, Becnel J J,Hazard E I. Taxonomy of phylum Microspora. Crit Rev Microbiol,1992,18 (5-6): 285-395.
[48]Franzen C,Muller A. Molecular techniques for detection, species differentiation, and phylogenetic analysis of microsporidia. Clin Microbiol Rev,1999,12 (2):243-285.
[49]Vossbrinck C R, Maddox J V, Friedman S, et al. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature,1987,326 (6111):411-414.
[50]Keeling P J. Congruent evidence from a-tubulin and (3-tubulin gene phylogenies for a zygomycete origin of microsporidia. Fungal Genet Biol,2003,38 (3):298-309.
[51]Keeling P J, Luker M A,Palmer J D. Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi. Mol Biol Evol,2000,17 (1):23-31.
[52]Cavalier-Smith T. A revised six-kingdom system of life. Biol Rev,1998,73 (3):203-266.
[53]Thomarat F, Vivares C P,Gouy M. Phylogenetic analysis of the complete genome sequence of Encephalitozoon cuniculi supports the fungal origin of microsporidia and reveals a high frequency of fast-evolving genes. J Mol Evol, 2004,59 (6):780-791.
[54]Leipe D D, Gunderson J H, Nerad T A, et al. Small subunit ribosomal RNA+of Hexamita inflata and the quest for the first branch in the eukaryotic tree. Mol Biochem Parasitol,1993,59 (1):41-48.
[55]Baldauf S L, Roger A J, Wenk-Siefert 1, et al. A kingdom-level phylogeny of eukaryotes based on combined protein data. Science,2000,290 (5493):972-977.
[56]Lee S C, Corradi N, Byrnes E J, et al. Microsporidia evolved from ancestral sexual fungi. Curr Biol,2008,18 (21):1675-1679.
[57]Gill E E,Fast N M. Assessing the microsporidia-fungi relationship:Combined phylogenetic analysis of eight genes. Gene,2006,375 103-109.
[58]Germot A, Philippe H,Le Guyader H. Evidence for loss of mitochondria in Microsporidia from a mitochondrial-type HSP70 in Nosema locustae. Mol Biochem Parasitol,1997,87 (2):159-168.
[59]Hirt R P, Healy B, Vossbrinck C R et al. A mitochondrial Hsp70 orthologue in Vairimorpha necatrix:molecular evidence that microsporidia once contained mitochondria. Curr Biol,1997,7(12):995-998.
[60]James T Y, Kauff F, Schoch C L, et al. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature,2006,443 (7113):818-822.
[61]Biderre C, Duffieux F, Peyretaillade E, et al. Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi. Gene,1997,191 (1):39-45.
[62]Brugere J F, Cornillot E, Metenier G, et al. Encephalitozoon cuniculi (Microspora) genome:physical map and evidence for telomere-associated rDNA units on all chromosomes. Nucleic Acids Res,2000,28 (10):2026-2033.
[63]Peyret P, Katinka M D, Duprat S, et al. Sequence and analysis of chromosome I of the amitochondriate intracellular parasite Encephalitozoon cuniculi (Microspora). Genome Res,2001,11 (2):198-207.
[64]Slamovits C H, Fast N M, Law J S, et al. Genome compaction and stability in microsporidian intracellular parasites. Curr Biol,2004,14 (10):891-896.
[65]Williams BAP, Lee R C H, Becnel J J, et al. Genome sequence surveys of Brachiola algerae and Edhazardia aedis reveal microsporidia with low gene densities. BMC Genomics,2008,9(1):200.
[66]Akiyoshi D E, Morrison H G, Lei S, et al. Genomic survey of the non-cultivatable opportunistic human pathogen, Enterocytozoon bieneusi. PLoS Pathog,2009,5 (1):e1000261.
[67]Cornman R S, Chen Y P, Schatz M C, et al. Genomic analyses of the microsporidian Nosema ceranae, an emergent pathogen of honey bees. PLoS Pathog,2009,5 (6):e1000466.
[68]Altschul S F, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol,1990,215 (3):403-410.
[69]Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res,1997,25 (24):4876-4882.
[70]Tamura K, Dudley J, Nei M, et al. MEGA4:Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol,2007,24 (8):1596-1599.
[71]Guindon S,Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology,2003,52 (5):696-704.
[72]Podell S,Gaasterland T. DarkHorse:a method for genome-wide prediction of horizontal gene transfer. Genome Biol,2007,8(2):R16.
[73]Ho J W K, Adams C E, Lew J B, et al. SeqVis:Visualization of compositional heterogeneity in large alignments of nucleotides. Bioinformatics,2006,22 (17):2162-2163.
[74]Rice P, Longden I,Bleasby A. EMBOSS:the European molecular biology open software suite. Trends Genet, 2000,16 (6):276-277.
[75]Zhang Z, Li J, Zhao X Q, et al. KaKs_Calculator:calculating Ka and Ks through model selection and model averaging. BMC Bioinformatics,2006,4 (4):259-263.
[76]Yang Z. PAML:a program package for phylogenetic analysis by maximum likelihood. Bioinformatics,1997,13 (5):555-556.
[77]Amano K, Nakamura H, Ichikawa H. Self-Organizing Clustering:A novel non-hierarchical method for clustering large amount of DNA sequences. Genome Informatics,2003,14 575-576.
[78]Wright F. The'effective number of codons'used in a gene. Gene,1990,87 (1):23-29.
[79]Hirano T, Kinoshita N, Morikawa K, et al. Snap helix with knob and hole:essential repeats in S. pombe nuclear protein nuc~2+Cell,1990,60 (2):319-328.
[80]Lamb J R, Tugendreich S,Hieter P. Tetratrico peptide repeat interactions:to TPR or not to TPR? Trends Biochem Sci,1995,20 (7):257-259.
[81]Sikorski R S, Boguski M S, Goebl M, et al. A repeating amino acid motif in CDC 23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell,1990,60 (2):307-317.
[82]Blatch GL,L ssle M. The tetratricopeptide repeat:a structural motif mediating protein-protein interactions. Bioessays,1999,21 (11):932-939.
[83]Gatto G J, Geisbrecht B V, Gould S J, et al. Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol,2000,7 (12):1091-1095.
[84]Lapouge K, Smith S J M, Walker P A, et al. Structure of the TPR domain of p67phox in complex with Rac-GTP. Mol Cell,2000,6 (4):899-907.
[85]Silva J C.Kidwell M G Horizontal transfer and selection in the evolution of P elements. Mol Biol Evol,2000, 17 (10):1542-1557.
[86]Diao X, Freeling M,Lisch D. Horizontal transfer of a plant transposon. PLoS Biol,2006,4 (1):e5.
[87]Page RDM. Tree View:an application to display phylogenetic trees on personal computers. Bioinformatics, 1996,12 (4):357-358.
[88]Muto A,Osawa S. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci US A,1987,84(1):166-169.
[89]Ciccarelli F D, Doerks T, von Mering C, et al. Toward automatic reconstruction of a highly resolved tree of life. Science,2006,311 (5765):1283-1287.
[90]Nakai K,Horton P. PSORT:a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci,1999,24 (1):34-35.
[91]Emanuelsson O, Nielsen H, Brunak S, et al. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol,2000,300 (4):1005-1016.
[92]Claros M G,Vincens P. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem,1996,241 (3):779-786.
[93]Edgar R C. MUSCLE:multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 2004,32 (5):1792-1797.
[94]Katoh K, Misawa K, Kuma K, et al. MAFFT:a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res,2002,30 (14):3059-3066.
[95]Subramanian A R, Kaufmann M,Morgenstern B. DIALIGN-TX:greedy and progressive approaches for segment-based multiple sequence alignment. Algorithms Mol Biol,2008,3 (1):6.
[96]Woodcock S, Mornon J P,Henrissat B. Detection of secondary structure elements in proteins by hydrophobic cluster analysis. Protein Eng,1992,5 (7):629-635.
[97]Comeron J M. K-Estimator:calculation of the number of nucleotide substitutions per site and the confidence intervals. Bioinformatics,1999,15 (9):763-764.
[98]Abascal F, Zardoya R,Posada D. ProtTest:selection of best-fit models of protein evolution. Bioinformatics, 2005,21 (9):2104-2105.
[99]Frealle E, Noel C, Nolard N, et al. Manganese superoxide dismutase based phytogeny of pathogenic fungi. Mol Phylogenet Evol,2006,41 (1):28-39.
[100]Wintjens R, Noel C, May A C, et al. Specificity and phenetic relationships of iron-and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons. J Biol Chem,2004,279 (10):9248-9254.
[101]Gaboriaud C, Bissery V, Benchetrit T, et al. Hydrophobic cluster analysis:an efficient new way to compare and analyse amino acid sequences. FEBS Lett,1987,224 (1):149-155.
[102]Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol,1980,16 (2):111-120.
[103]Ababneh F, Jermiin L S, Ma C, et al. Matched-pairs tests of homogeneity with applications to homologous nucleotide sequences. Bioinformatics,2006,22 (10):1225-1231.
[104]Bergsten J. A review of long-branch attraction. Cladistics,2005,21 (2):163-193.
[105]Bergsten J,Miller K B. Acilius phylogeny (Coleoptera:Dytiscidae), problems with long-branch attraction and morphological intersexual coevlution [Meeting Abstract], Cladistics,2004,2076-77.
[106]Hampl V, Cepicka I, Flegr J, et al. Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree. Mol Phylogenet Evol,2004,32 (3):711-723.
[107]Parker M W,Blake C C. Iron-and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett,1988,229 (2):377-382.
[108]Jackson S M,Cooper J B. An analysis of structural similarity in the iron and manganese superoxide dismutases based on known structures and sequences. Biometals,1998,11 (2):159-173.
[109]Viscogliosi E, Delgado-Viscogliosi P, Gerbod D, et al. Cloning and expression of an iron-containing superoxide dismutase in the parasitic protist, Trichomonas vaginalis. FEMS Microbiol Lett,1998,161 (1):115-123.
[110]Takao M, Yasui A,Oikawa A. Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum. J Biol Chem,1991,266 (22):14151-14154.
[111]Barra D, Schinina M E, Bossa F, et al. A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis. J Biol Chem,1990,265 (29):17680-17687.
[112]Lah M S, Dixon M M, Partridge K A, et al. Structure-function in Escherichia coli iron superoxide dismutase: comparisons with the manganese enzyme from Thermus thermophilus. Biochemistry,1995,34 (5):1646-1660.
[113]Borgstahl G E, Parge H E, Hickey M J, et al. The structure of human mitochondrial manganese superoxide dismutase reveals anovel tetrameric interface of two 4-helix bundles. Cell,1992,71 (1):107-118.
[114]Cooper J B, Mclntyre K, Badasso M O, et al. X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions. J Mol Biol,1995,246 (4):531-544.
[115]Blackman L M, Mitchell H J,Hardham A R. Characterisation of manganese superoxide dismutase from Phytophthora nicotianae. Mycol Res,2005,109 (Pt 10):1171-1183.
[116]Natvig D O, Sylvester K, Dvorachek W H, et al. Superoxide dismutases and catalases. Biochemistry and molecular Biology:In Brambl, R., Marzluf, GA. (eds),1996.
[117]Palma J M, Lopez-Huertas E, Corpas F J, et al. Peroxisomal manganese superoxide dismutase:Purification and properties of the isozyme from pea leaves. Physiol Plant,1998,104 (4):720-726.
[118]Alscher R G Erturk N.Heath L S. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot,2002,53 (372):1331-1341.
[119]Weisiger R A,Fridovich I. Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization. J Biol Chem,1973,248 (13):4793-4796.
[120]Zhu D,Scandalios J G. Maize mitochondrial manganese superoxide dismutases are encoded by a differentially expressed multigene family. ProcNatl Acad Sci USA,1993,90 (20):9310-9314.
[121]Lynch M,Kuramitsu H. Expression and role of superoxide dismutases (SOD) in pathogenic bacteria. Microbes Infect,2000,2 (10):1245-1255.
[122]Fang G C, Hanau R M,Vaillancourt L J. The SOD2 gene, encoding a manganese-type superoxide dismutase, is up-regulated during conidiogenesis in the plant-pathogenic fungus Colletotrichum graminicola. Fungal Genet Biol, 2002,36 (2):155-165.
[123]Bagnoli F, Capuana M,Raccfii M L. Developmental changes of catalase and superoxide dismutase isoenzymes in zygotic and somatic embryos of horse chestnut. J Plant Physiol,1998,25 (8):909-913.
[124]Regelsberger G, Atzenhofer W, Ruker F, et al. Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120. J Biol Chem,2002,277 (46):43615-43622.
[125]Kliebenstein D J, Monde R A,Last R L. Superoxide dismutase in Arabidopsis:an eclectic enzyme family with disparate regulation and protein localization. Plant Physiol,1998,118 (2):637-650.
[126]Babitha M P, Bhat S G, Prakash H S, et al. Differential induction of superoxide dismutase in downy mildew-resistant and-susceptible genotypes of pearl millet. Plant Pathol,2002,51 (4):480-486.
[127]Van Camp W, Inze D,Van Montagu M. The regulation and function of tobacco superoxide dismutases. Free Radic Biol Med,1997,23 (3):515-520.
[128]Brouwer M, Brouwer T H, Grater W, et al. The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods. Biochemistry,1997,36 (43):13381-13388.
[129]Hunter T, Bannister W H.Hunter G J. Cloning, expression, and characterization of two manganese superoxide dismutases from Caenorhabditis elegans. J Biol Chem,1997,272 (45):28652-28659.
[130]Bagnoli F, Giannino D, Caparrini S, et al. Molecular cloning, characterisation and expression of a manganese superoxide dismutase gene from peach (Prunuspersica [L.] Batsch). Mol Genet Genomics,2002,267 (3):321-328.
[131]Scandalios J G Oxygen stress and superoxide dismutases. Plant Physiol,1993,101 (1):7-12.
[132]Marklund S L. Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species. Biochem J,1984,222 (3):649-655.
[133]Ohno S. Evolution by Gene Duplication. Springer Verlag Press, New York:1970.
[134]Irving J A, Pike R N, Lesk A M, et al. Phylogeny of the serpin superfamily:implications of patterns of amino acid conservation for structure and function. Genome Res,2000,10 (12):1845-1864.
[135]Steenbakkers P J M, Irving J A, Harhangi H R, et al. A serpin in the cellulosome of the anaerobic fungus Piromyces sp. strain E2. Mycol Res,2008,112 (8):999-1006.
[136]Zhou Q, Zhang G, Zhang Y, et al. On the origin of new genes in Drosophila. Genome Res,2008,18 (9): 1446-1455.
[137]Li R, Ye J, Li S, etal. ReAS:Recovery of ancestral sequences for transposable elements from the unassembled reads of a whole genome shotgun. PLoS Comput Biol,2005,1 (4):e43.
[138]Edgar R C,Myers E W. PILER:identification and classification of genomic repeats. Bioinformatics,2005,21 (1):152-158.
[139]Price A L, Jones N C,Pevzner P A. De novo identification of repeat families in large genomes. Bioinformatics, 2005,21(1):351-358.
[140]Xu Z,Wang H. LTR_FINDER:an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res 2007,35 (2):W265-W268.
[141]Rozas J, Sanchez-DelBarrio J C, Messeguer X, et al. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics,2003,19 (18):2496-2497.
[142]Xu J, Pan Q Fang L, et al. The varying microsporidian genome:existence of long-terminal repeat retrotransposon in domesticated silkworm parasite Nosema bombycis. Int J Parasitol,2006,36 (9):1049-1056.
[143]Laha T, Loukas A, Smyth D J, et al. The fugitive LTR retrotransposon from the genome of the human blood fluke, Schistosoma mansoni. Int J Parasitol,2004,34 (12):1365-1375.
[144]Nei M. Molecular Evolutionary Genetics. Columbia Univ. Press, New York:In Nevo, E., Beiles, A., Schlomo, R., Ben Mani, G.S. (eds),1987.
[145]Morrison H Q McArthur A G, Gillin F D, et al. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science,2007,317 (5846):1921-1926.
[146]Petrov D A. Evolution of genome size:new approaches to an old problem. Trends Genet,2001,17 (1):23-28.
[147]Volff J N, Lehrach H, Reinhardt R, et al. Retroelement dynamics and a novel type of chordate retrovirus-like element in the miniature genome of the tunicate Oikopleura dioica. Mol Biol Evol,2004,21 (11):2022-2033.
[2]Ochman H, Lawrence J G,Groisman E A. Lateral gene transfer and the nature of bacterial innovation. Nature, 2000,405 (6784):299-304.
[3]Keeling P J,Palmer J D. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet,2008,9 (8):605-618.
[4]Woese C. The universal ancestor. Proc Natl Acad Sci USA,1998,95 (12):6854-6859.
[5]Doolittle W F. Phylogenetic classification and the universal tree. Science,1999,284 (5423):2124-2128.
[6]Smets B F,Barkay T. Horizontal gene transfer:perspectives at a crossroads of scientific disciplines. Nat Rev Microbiol,2005,3 (9):675-678.
[7]Choi I G,Kim S H. Global extent of horizontal gene transfer. Proc Natl Acad Sci USA,2007,104 (11): 4489-4494.
[8]Ricard G, McEwan N R, Dutilh B E, et al. Horizontal gene transfer from Bacteria to rumen Ciliates indicates adaptation to their anaerobic, carbohydrates-rich environment. BMC Genomics,2006,7 (1):22.
[9]Syvanen M. Recent emergence of the modern genetic code:a proposal. Trends Genet,2002,18 (5):245-248.
[10]Jain R, Rivera M C,Lake.J A. Horizontal gene transfer among genomes:the complexity hypothesis. Proc Natl Acad Sci USA,1999,96 (7):3801-3806.
[11]Eisen J A. Horizontal gene transfer among microbial genomes:new insights from complete genome analysis. Curr Opin Genet Dev,2000,10 (6):606-611.
[12]Lawrence J G,Ochman H. Amelioration of Bacterial Genomes:Rates of Change and Exchange. J Mol Biol, 1997,44 (4):383-397.
[13]Mosig G Recombination and recombination-dependent DNA replication in bacteriophage T4. Annu Rev Genet, 1998,32(1):379-413.
[14]Jain R, Rivera M C, Moore J E, et al. Horizontal gene transfer accelerates genome innovation and evolution. Mol Biol Evol,2003,20 (10):1598-1602.
[15]Gogarten J P,Townsend J P. Horizontal gene transfer, genome innovation and evolution. Nat Rev Microbiol, 2005,3 (9):679-687.
[16]Martin W. Mosaic bacterial chromosomes:a challenge en route to a tree of genomes. Bioessays,1999,21 (2): 99-104.
[17]Griffith F. The signi cance of pneumococcal types. J Hyg,1928,27 113-159.
[18]Avery O T, McLeod C M,McCarthy M. Studies on the Chemical nature of the substance inducing transformation of pneumococcal types:Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med,1944,79 (2):137-158.
[19]McClintock B. The origin and behavior of mutable loci in maize. Proc Natl Acad Sci USA,1950,36 (6): 344-355.
[20]Lederberg J,Tatum E L. Novel genotypes in mixed cultures of biochemical mutants of bacteria, cold spring harb symp quant biol,1946,11 113-114.
[21]Ochiai K, Yamanaka T, Kimura K, et al. Inheritance of drug resistance (and its transfer) between Shigella strains and between Shigella and E. coli strains. Nihon Iji Shimpo 1861,1959,34
[22]Shapiro J A. Mutations caused by the insertion of genetic material into the galactose operon of Escherichia coli. J Mol Biol,1969,40 (1):93-105.
[23]Went F W. Parallel evolution. Taxon,1971,20 (2):197-226.
[24]Hiraizumi Y. Spontaneous recombination in Drosophila melanogaster males. Proc Natl Acad Sci U S A,1971, 68 (2):268-270.
[25]Green M M. Genetic instability in Drosophila melanogaster. De novo induction of putative insertion mutations. Proc Natl Acad Sci USA,1977,74 (8):3490-3493.
[26]Syvanen M. Cross-species gene transfer; implications for a new theory of evolution. J Theor Biol,1985,112 (2): 333-343.
[27]Ho M W, Ryan A,Cummins J. Cauliflower Mosaic Viral Promoter-A Recipe for Disaster? Microbial Ecology in Health and Disease,1999,11 (4):194-197.
[28]Gogarten P. Horizontal Gene Transfer:A New Paradigm for Biology. Esalen Center for Theory and Research Conference,2000,
[29]Bergthorsson U, Adams K L, Thomason B, et al. Widespread horizontal transfer of mitochondrial genes in flowering plants. Nature,2003,424 (6945):197-201.
[30]Rivera M C,Lake J A. The ring of life provides evidence for a genome fusion origin of eukaryotes. Nature,2004, 431 (7005):152-155.
[31]Bapteste E, Susko E, Leigh J, et al. Do orthologous gene phylogenies really support tree-thinking? BMC Evol Biol,2005,5(1):33.
[32]Huang J, Mullapudi N, Lancto C, et al. Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum. Genome Biol,2004,5 (11):R88.
[33]Loftus B, Anderson I, Davies R, et al. The genome of the protist parasite Entamoeba histolytica. Nature,2005, 433 (7028):865-868.
[34]Eichinger L, Pachebat J A, Glockner G, et al. The genome of the social amoeba Dictyostelium discoideum. Nature,2005,435 (7038):43-57.
[35]Berriman M, Ghedin E, Hertz-Fowler C, et al. The genome of the African trypanosome Trypanosoma brucei. Science,2005,309 (5733):416-422.
[36]Derelle E, Ferraz C, Rombauts S, et al. Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc Natl Acad Sci U S A,2006,103 (31):11647-11652.
[37]Carlton J M, Hirt R P, Silva J C, et al. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science,2007,315 (5809):207-212.
[38]Gladyshev E A, Meselson M,Arkhipova I R. Massive horizontal gene transfer in bdelloid rotifers. Science,2008, 320(5880):1210-1213.
[39]Winner M. Historic perspective on the microsporidia:expanding horizons. In:The microsporidia and microsporidiosis. Amer Society for Microbiology,1999.
[40]Larsson J I R. Identification of microsporidia. Acta protozoologica,1999,38 (3):161-197.
[41]Weber R, Bryan R T, Schwartz D A, et al. Human microsporidial infections. Clin Microbiol Rev,1994,7 (4): 426-461.
[42]Mathis A, Weber R,Deplazes P. Zoonotic potential of the microsporidia. Clin Microbiol Rev,2005,18 (3): 423-445.
[43]Visvesvara G S, Belloso M, Moura H, et al. Isolation of Nosema algerae from the cornea of an immunocompetent patient. J Eukaryot Microbiol,1999,46 (5):10S.
[44]Svenungsson B, Capraru T, Eveng rd B, et al. Intestinal microsporidiosis in a HIV-seronegative patient. Scand J Infect Dis,1998,30 (3):314-316.
[45]Lopez-Velez R, Turrientes M C, Garron C, et al. Microsporidiosis in travelers with diarrhea from the tropics. J Travel Med,1999,6 (4):223-227.
[46]Katinka M D, Duprat S, Cornillot E, et al. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature,2001,414 (6862):450-453.
[47]Sprague V, Becnel J J,Hazard E I. Taxonomy of phylum Microspora. Crit Rev Microbiol,1992,18 (5-6): 285-395.
[48]Franzen C,Muller A. Molecular techniques for detection, species differentiation, and phylogenetic analysis of microsporidia. Clin Microbiol Rev,1999,12 (2):243-285.
[49]Vossbrinck C R, Maddox J V, Friedman S, et al. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature,1987,326 (6111):411-414.
[50]Keeling P J. Congruent evidence from a-tubulin and (3-tubulin gene phylogenies for a zygomycete origin of microsporidia. Fungal Genet Biol,2003,38 (3):298-309.
[51]Keeling P J, Luker M A,Palmer J D. Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi. Mol Biol Evol,2000,17 (1):23-31.
[52]Cavalier-Smith T. A revised six-kingdom system of life. Biol Rev,1998,73 (3):203-266.
[53]Thomarat F, Vivares C P,Gouy M. Phylogenetic analysis of the complete genome sequence of Encephalitozoon cuniculi supports the fungal origin of microsporidia and reveals a high frequency of fast-evolving genes. J Mol Evol, 2004,59 (6):780-791.
[54]Leipe D D, Gunderson J H, Nerad T A, et al. Small subunit ribosomal RNA+of Hexamita inflata and the quest for the first branch in the eukaryotic tree. Mol Biochem Parasitol,1993,59 (1):41-48.
[55]Baldauf S L, Roger A J, Wenk-Siefert 1, et al. A kingdom-level phylogeny of eukaryotes based on combined protein data. Science,2000,290 (5493):972-977.
[56]Lee S C, Corradi N, Byrnes E J, et al. Microsporidia evolved from ancestral sexual fungi. Curr Biol,2008,18 (21):1675-1679.
[57]Gill E E,Fast N M. Assessing the microsporidia-fungi relationship:Combined phylogenetic analysis of eight genes. Gene,2006,375 103-109.
[58]Germot A, Philippe H,Le Guyader H. Evidence for loss of mitochondria in Microsporidia from a mitochondrial-type HSP70 in Nosema locustae. Mol Biochem Parasitol,1997,87 (2):159-168.
[59]Hirt R P, Healy B, Vossbrinck C R et al. A mitochondrial Hsp70 orthologue in Vairimorpha necatrix:molecular evidence that microsporidia once contained mitochondria. Curr Biol,1997,7(12):995-998.
[60]James T Y, Kauff F, Schoch C L, et al. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature,2006,443 (7113):818-822.
[61]Biderre C, Duffieux F, Peyretaillade E, et al. Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi. Gene,1997,191 (1):39-45.
[62]Brugere J F, Cornillot E, Metenier G, et al. Encephalitozoon cuniculi (Microspora) genome:physical map and evidence for telomere-associated rDNA units on all chromosomes. Nucleic Acids Res,2000,28 (10):2026-2033.
[63]Peyret P, Katinka M D, Duprat S, et al. Sequence and analysis of chromosome I of the amitochondriate intracellular parasite Encephalitozoon cuniculi (Microspora). Genome Res,2001,11 (2):198-207.
[64]Slamovits C H, Fast N M, Law J S, et al. Genome compaction and stability in microsporidian intracellular parasites. Curr Biol,2004,14 (10):891-896.
[65]Williams BAP, Lee R C H, Becnel J J, et al. Genome sequence surveys of Brachiola algerae and Edhazardia aedis reveal microsporidia with low gene densities. BMC Genomics,2008,9(1):200.
[66]Akiyoshi D E, Morrison H G, Lei S, et al. Genomic survey of the non-cultivatable opportunistic human pathogen, Enterocytozoon bieneusi. PLoS Pathog,2009,5 (1):e1000261.
[67]Cornman R S, Chen Y P, Schatz M C, et al. Genomic analyses of the microsporidian Nosema ceranae, an emergent pathogen of honey bees. PLoS Pathog,2009,5 (6):e1000466.
[68]Altschul S F, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol,1990,215 (3):403-410.
[69]Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res,1997,25 (24):4876-4882.
[70]Tamura K, Dudley J, Nei M, et al. MEGA4:Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol,2007,24 (8):1596-1599.
[71]Guindon S,Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology,2003,52 (5):696-704.
[72]Podell S,Gaasterland T. DarkHorse:a method for genome-wide prediction of horizontal gene transfer. Genome Biol,2007,8(2):R16.
[73]Ho J W K, Adams C E, Lew J B, et al. SeqVis:Visualization of compositional heterogeneity in large alignments of nucleotides. Bioinformatics,2006,22 (17):2162-2163.
[74]Rice P, Longden I,Bleasby A. EMBOSS:the European molecular biology open software suite. Trends Genet, 2000,16 (6):276-277.
[75]Zhang Z, Li J, Zhao X Q, et al. KaKs_Calculator:calculating Ka and Ks through model selection and model averaging. BMC Bioinformatics,2006,4 (4):259-263.
[76]Yang Z. PAML:a program package for phylogenetic analysis by maximum likelihood. Bioinformatics,1997,13 (5):555-556.
[77]Amano K, Nakamura H, Ichikawa H. Self-Organizing Clustering:A novel non-hierarchical method for clustering large amount of DNA sequences. Genome Informatics,2003,14 575-576.
[78]Wright F. The'effective number of codons'used in a gene. Gene,1990,87 (1):23-29.
[79]Hirano T, Kinoshita N, Morikawa K, et al. Snap helix with knob and hole:essential repeats in S. pombe nuclear protein nuc~2+Cell,1990,60 (2):319-328.
[80]Lamb J R, Tugendreich S,Hieter P. Tetratrico peptide repeat interactions:to TPR or not to TPR? Trends Biochem Sci,1995,20 (7):257-259.
[81]Sikorski R S, Boguski M S, Goebl M, et al. A repeating amino acid motif in CDC 23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell,1990,60 (2):307-317.
[82]Blatch GL,L ssle M. The tetratricopeptide repeat:a structural motif mediating protein-protein interactions. Bioessays,1999,21 (11):932-939.
[83]Gatto G J, Geisbrecht B V, Gould S J, et al. Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol,2000,7 (12):1091-1095.
[84]Lapouge K, Smith S J M, Walker P A, et al. Structure of the TPR domain of p67phox in complex with Rac-GTP. Mol Cell,2000,6 (4):899-907.
[85]Silva J C.Kidwell M G Horizontal transfer and selection in the evolution of P elements. Mol Biol Evol,2000, 17 (10):1542-1557.
[86]Diao X, Freeling M,Lisch D. Horizontal transfer of a plant transposon. PLoS Biol,2006,4 (1):e5.
[87]Page RDM. Tree View:an application to display phylogenetic trees on personal computers. Bioinformatics, 1996,12 (4):357-358.
[88]Muto A,Osawa S. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci US A,1987,84(1):166-169.
[89]Ciccarelli F D, Doerks T, von Mering C, et al. Toward automatic reconstruction of a highly resolved tree of life. Science,2006,311 (5765):1283-1287.
[90]Nakai K,Horton P. PSORT:a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci,1999,24 (1):34-35.
[91]Emanuelsson O, Nielsen H, Brunak S, et al. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol,2000,300 (4):1005-1016.
[92]Claros M G,Vincens P. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem,1996,241 (3):779-786.
[93]Edgar R C. MUSCLE:multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 2004,32 (5):1792-1797.
[94]Katoh K, Misawa K, Kuma K, et al. MAFFT:a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res,2002,30 (14):3059-3066.
[95]Subramanian A R, Kaufmann M,Morgenstern B. DIALIGN-TX:greedy and progressive approaches for segment-based multiple sequence alignment. Algorithms Mol Biol,2008,3 (1):6.
[96]Woodcock S, Mornon J P,Henrissat B. Detection of secondary structure elements in proteins by hydrophobic cluster analysis. Protein Eng,1992,5 (7):629-635.
[97]Comeron J M. K-Estimator:calculation of the number of nucleotide substitutions per site and the confidence intervals. Bioinformatics,1999,15 (9):763-764.
[98]Abascal F, Zardoya R,Posada D. ProtTest:selection of best-fit models of protein evolution. Bioinformatics, 2005,21 (9):2104-2105.
[99]Frealle E, Noel C, Nolard N, et al. Manganese superoxide dismutase based phytogeny of pathogenic fungi. Mol Phylogenet Evol,2006,41 (1):28-39.
[100]Wintjens R, Noel C, May A C, et al. Specificity and phenetic relationships of iron-and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons. J Biol Chem,2004,279 (10):9248-9254.
[101]Gaboriaud C, Bissery V, Benchetrit T, et al. Hydrophobic cluster analysis:an efficient new way to compare and analyse amino acid sequences. FEBS Lett,1987,224 (1):149-155.
[102]Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol,1980,16 (2):111-120.
[103]Ababneh F, Jermiin L S, Ma C, et al. Matched-pairs tests of homogeneity with applications to homologous nucleotide sequences. Bioinformatics,2006,22 (10):1225-1231.
[104]Bergsten J. A review of long-branch attraction. Cladistics,2005,21 (2):163-193.
[105]Bergsten J,Miller K B. Acilius phylogeny (Coleoptera:Dytiscidae), problems with long-branch attraction and morphological intersexual coevlution [Meeting Abstract], Cladistics,2004,2076-77.
[106]Hampl V, Cepicka I, Flegr J, et al. Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree. Mol Phylogenet Evol,2004,32 (3):711-723.
[107]Parker M W,Blake C C. Iron-and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett,1988,229 (2):377-382.
[108]Jackson S M,Cooper J B. An analysis of structural similarity in the iron and manganese superoxide dismutases based on known structures and sequences. Biometals,1998,11 (2):159-173.
[109]Viscogliosi E, Delgado-Viscogliosi P, Gerbod D, et al. Cloning and expression of an iron-containing superoxide dismutase in the parasitic protist, Trichomonas vaginalis. FEMS Microbiol Lett,1998,161 (1):115-123.
[110]Takao M, Yasui A,Oikawa A. Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum. J Biol Chem,1991,266 (22):14151-14154.
[111]Barra D, Schinina M E, Bossa F, et al. A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis. J Biol Chem,1990,265 (29):17680-17687.
[112]Lah M S, Dixon M M, Partridge K A, et al. Structure-function in Escherichia coli iron superoxide dismutase: comparisons with the manganese enzyme from Thermus thermophilus. Biochemistry,1995,34 (5):1646-1660.
[113]Borgstahl G E, Parge H E, Hickey M J, et al. The structure of human mitochondrial manganese superoxide dismutase reveals anovel tetrameric interface of two 4-helix bundles. Cell,1992,71 (1):107-118.
[114]Cooper J B, Mclntyre K, Badasso M O, et al. X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions. J Mol Biol,1995,246 (4):531-544.
[115]Blackman L M, Mitchell H J,Hardham A R. Characterisation of manganese superoxide dismutase from Phytophthora nicotianae. Mycol Res,2005,109 (Pt 10):1171-1183.
[116]Natvig D O, Sylvester K, Dvorachek W H, et al. Superoxide dismutases and catalases. Biochemistry and molecular Biology:In Brambl, R., Marzluf, GA. (eds),1996.
[117]Palma J M, Lopez-Huertas E, Corpas F J, et al. Peroxisomal manganese superoxide dismutase:Purification and properties of the isozyme from pea leaves. Physiol Plant,1998,104 (4):720-726.
[118]Alscher R G Erturk N.Heath L S. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot,2002,53 (372):1331-1341.
[119]Weisiger R A,Fridovich I. Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization. J Biol Chem,1973,248 (13):4793-4796.
[120]Zhu D,Scandalios J G. Maize mitochondrial manganese superoxide dismutases are encoded by a differentially expressed multigene family. ProcNatl Acad Sci USA,1993,90 (20):9310-9314.
[121]Lynch M,Kuramitsu H. Expression and role of superoxide dismutases (SOD) in pathogenic bacteria. Microbes Infect,2000,2 (10):1245-1255.
[122]Fang G C, Hanau R M,Vaillancourt L J. The SOD2 gene, encoding a manganese-type superoxide dismutase, is up-regulated during conidiogenesis in the plant-pathogenic fungus Colletotrichum graminicola. Fungal Genet Biol, 2002,36 (2):155-165.
[123]Bagnoli F, Capuana M,Raccfii M L. Developmental changes of catalase and superoxide dismutase isoenzymes in zygotic and somatic embryos of horse chestnut. J Plant Physiol,1998,25 (8):909-913.
[124]Regelsberger G, Atzenhofer W, Ruker F, et al. Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120. J Biol Chem,2002,277 (46):43615-43622.
[125]Kliebenstein D J, Monde R A,Last R L. Superoxide dismutase in Arabidopsis:an eclectic enzyme family with disparate regulation and protein localization. Plant Physiol,1998,118 (2):637-650.
[126]Babitha M P, Bhat S G, Prakash H S, et al. Differential induction of superoxide dismutase in downy mildew-resistant and-susceptible genotypes of pearl millet. Plant Pathol,2002,51 (4):480-486.
[127]Van Camp W, Inze D,Van Montagu M. The regulation and function of tobacco superoxide dismutases. Free Radic Biol Med,1997,23 (3):515-520.
[128]Brouwer M, Brouwer T H, Grater W, et al. The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods. Biochemistry,1997,36 (43):13381-13388.
[129]Hunter T, Bannister W H.Hunter G J. Cloning, expression, and characterization of two manganese superoxide dismutases from Caenorhabditis elegans. J Biol Chem,1997,272 (45):28652-28659.
[130]Bagnoli F, Giannino D, Caparrini S, et al. Molecular cloning, characterisation and expression of a manganese superoxide dismutase gene from peach (Prunuspersica [L.] Batsch). Mol Genet Genomics,2002,267 (3):321-328.
[131]Scandalios J G Oxygen stress and superoxide dismutases. Plant Physiol,1993,101 (1):7-12.
[132]Marklund S L. Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species. Biochem J,1984,222 (3):649-655.
[133]Ohno S. Evolution by Gene Duplication. Springer Verlag Press, New York:1970.
[134]Irving J A, Pike R N, Lesk A M, et al. Phylogeny of the serpin superfamily:implications of patterns of amino acid conservation for structure and function. Genome Res,2000,10 (12):1845-1864.
[135]Steenbakkers P J M, Irving J A, Harhangi H R, et al. A serpin in the cellulosome of the anaerobic fungus Piromyces sp. strain E2. Mycol Res,2008,112 (8):999-1006.
[136]Zhou Q, Zhang G, Zhang Y, et al. On the origin of new genes in Drosophila. Genome Res,2008,18 (9): 1446-1455.
[137]Li R, Ye J, Li S, etal. ReAS:Recovery of ancestral sequences for transposable elements from the unassembled reads of a whole genome shotgun. PLoS Comput Biol,2005,1 (4):e43.
[138]Edgar R C,Myers E W. PILER:identification and classification of genomic repeats. Bioinformatics,2005,21 (1):152-158.
[139]Price A L, Jones N C,Pevzner P A. De novo identification of repeat families in large genomes. Bioinformatics, 2005,21(1):351-358.
[140]Xu Z,Wang H. LTR_FINDER:an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res 2007,35 (2):W265-W268.
[141]Rozas J, Sanchez-DelBarrio J C, Messeguer X, et al. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics,2003,19 (18):2496-2497.
[142]Xu J, Pan Q Fang L, et al. The varying microsporidian genome:existence of long-terminal repeat retrotransposon in domesticated silkworm parasite Nosema bombycis. Int J Parasitol,2006,36 (9):1049-1056.
[143]Laha T, Loukas A, Smyth D J, et al. The fugitive LTR retrotransposon from the genome of the human blood fluke, Schistosoma mansoni. Int J Parasitol,2004,34 (12):1365-1375.
[144]Nei M. Molecular Evolutionary Genetics. Columbia Univ. Press, New York:In Nevo, E., Beiles, A., Schlomo, R., Ben Mani, G.S. (eds),1987.
[145]Morrison H Q McArthur A G, Gillin F D, et al. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science,2007,317 (5846):1921-1926.
[146]Petrov D A. Evolution of genome size:new approaches to an old problem. Trends Genet,2001,17 (1):23-28.
[147]Volff J N, Lehrach H, Reinhardt R, et al. Retroelement dynamics and a novel type of chordate retrovirus-like element in the miniature genome of the tunicate Oikopleura dioica. Mol Biol Evol,2004,21 (11):2022-2033.