Comparative Analysis Of Completely Sequenced Genomes

快速增长的细菌、古生菌和真核生物基因组全序列数据库（到2010年初已有1100多个基因组，还有更多的基因组正在开发中）为基因组研究创造了新的机遇和新的挑战。在过去的一年里，我们进行了几项研究，利用基因组信息建立基因组进化和功能的基本原则。特别是，我们进行了全面的比较分析，真核细胞核质大DNA病毒（NCLDV），包括构建的集群的orthopathic基因和重建的病毒基因组进化。NCLDV包含感染多种真核宿主的明显单系类病毒。最近在分离新病毒和基因组测序方面的进展导致NCLDV多样性的大幅扩展，从而为比较基因组分析提供了额外的机会，并需要对病毒基因进行全面分类。对6个科45株NCLDV的基因组编码蛋白序列进行了比较分析，以确定其基因簇。使用先前开发的计算方法进行同源性鉴定，鉴定了1445个核质病毒同源群（NCVOG），其中177个代表一个以上的NCLDV家族。NCVOG是人工策划和注释的，可以用作新NCLDV基因组的功能注释和进化分析的计算平台。NCLDV进化的最大似然重建产生了一组47个保守基因，这些基因可能存在于这类真核病毒的共同祖先的基因组中。该重建的祖先基因组对重建过程的参数是稳健的，因此可能准确地反映祖先NCLDV的基因核心，表明该病毒编码复杂的复制、表达和形态发生机制，使其相对独立于宿主细胞功能。NCVOG是一个灵活和可扩展的平台，用于新表征的NCLDV的基因组分析和功能注释。采用NCVOG的进化重建指向复杂的祖先病毒。 在另一个项目中，我们研究了细菌中I型毒素-抗毒素系统的丰度和分布，目的是寻找新的候选者和发现新的家族。小分子疏水蛋白的合成被小RNA（sRNA）抑制，称为I型毒素-抗毒素模块，首先在质粒上发现，它们调节质粒的稳定性，但随后在一些细菌染色体上发现。我们使用了详尽的PSI-BLAST和TBLASTIC搜索774个细菌基因组，以确定同源的已知I型毒素。这些搜索大大扩展了预测的I型毒素的集合，揭示了Ldr和Fst毒素的同源性，并表明I型毒素-抗毒素位点不通过水平基因转移传播。为了发现新的I型毒素抗毒素系统，我们开发了一套搜索参数的基础上的特征，包括存在的串联重复序列和集群的收费和庞大的氨基酸在C-末端的短蛋白质含有预测的跨膜区域。我们检测了三个预测的毒素从肠出血性大肠杆菌和枯草芽孢杆菌的sRNA，并表明，两个各自的蛋白质确实是有毒的过度表达时。我们还证明了RNA折叠的局部自由能最小值可以用来检测sRNA基因的位置。我们的研究结果表明，I型毒素-抗毒素模块在细菌中的分布比以前认识到的要广泛得多。 在一项单独的比较基因组研究中，我们探索了无内含子和含内含子的哺乳动物基因的表达和进化的不同模式。比较无内含子和含内含子的哺乳动物基因的表达水平、宽度和进化速率表明，无内含子基因的表达水平较低，倾向于组织特异性，并且进化速度明显快于剪接基因。相比之下，不受可检测的选择性剪接的单态剪接基因和多态性选择性剪接基因显示出相似的统计学上不可区分的表达和进化模式。选择性剪接在古老基因中最常见，而无内含子基因似乎起源相对较近。这些结果意味着基因表达的不同阶段之间的紧密耦合，特别是转录，剪接和转录本的核胞质转运，并表明无内含子基因的形成是动物中新的组织特异性功能进化的重要途径。

The rapidly growing database of completely sequenced genomes of bacteria, archaea and eukaryotes (over 1100 genomes available by the beginning of 2010 and many more in progress) creates both new opportunities and new challenges for genome research. During the last year, we performed several studies that took advantage of the genomic information to establish fundamental principles of genome evolution and function. In particular, we performed a comprehensive comparative analysis of eukaryotic nucleo-cytoplasmic large DNA viruses (NCLDV) including the construction of clusters of orthologous genes and reconstruction of viral genome evolution. The NCLDV comprise an apparently monophyletic class of viruses that infect a broad variety of eukaryotic hosts. Recent progress in isolation of new viruses and genome sequencing resulted in a substantial expansion of the NCLDV diversity, resulting in additional opportunities for comparative genomic analysis, and a demand for a comprehensive classification of viral genes. A comprehensive comparison of the protein sequences encoded in the genomes of 45 NCLDV belonging to 6 families was performed in order to delineate cluster of orthologous viral genes. Using previously developed computational methods for orthology identification, 1445 Nucleo-Cytoplasmic Virus Orthologous Groups (NCVOGs) were identified of which 177 are represented in more than one NCLDV family. The NCVOGs were manually curated and annotated and can be used as a computational platform for functional annotation and evolutionary analysis of new NCLDV genomes. A maximum-likelihood reconstruction of the NCLDV evolution yielded a set of 47 conserved genes that were probably present in the genome of the common ancestor of this class of eukaryotic viruses. This reconstructed ancestral gene set is robust to the parameters of the reconstruction procedure and so is likely to accurately reflect the gene core of the ancestral NCLDV, indicating that this virus encoded a complex machinery of replication, expression and morphogenesis that made it relatively independent from host cell functions. The NCVOGs are a flexible and expandable platform for genome analysis and functional annotation of newly characterized NCLDV. Evolutionary reconstructions employing NCVOGs point to complex ancestral viruses. In another project, we investigated the abundance and distribution of type I toxin-antitoxin systems in bacteria with the goals of searching for new candidates and discovering novel families. Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin-antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin-antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin-antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin-antitoxin modules are much more widely distributed among bacteria than previously appreciated. In a separate comparative genomic study we explored distinct patterns of expression and evolution of intronless and intron-containing mammalian genes. Comparison of expression levels and breadth and evolutionary rates of intronless and intron-containing mammalian genes shows that intronless genes are expressed at lower levels, tend to be tissue specific, and evolve significantly faster than spliced genes. By contrast, monomorphic spliced genes that are not subject to detectable alternative splicing and polymorphic alternatively spliced genes show similar statistically indistinguishable patterns of expression and evolution. Alternative splicing is most common in ancient genes, whereas intronless genes appear to have relatively recent origins. These results imply tight coupling between different stages of gene expression, in particular, transcription, splicing, and nucleocytosolic transport of transcripts, and suggest that formation of intronless genes is an important route of evolution of novel tissue-specific functions in animals.