An automated homology-based approach for identifying transposable elements

Background  

Transposable elements (TEs) are mobile sequences found in nearly all eukaryotic genomes. They have the ability to move and replicate within a genome, often influencing genome evolution and gene expression. The identification of TEs is an important part of every genome project. The number of sequenced genomes is rapidly rising, and the need to identify TEs within them is also growing. The ability to do this automatically and effectively in a manner similar to the methods used for genes is of increasing importance. There exist many difficulties in identifying TEs, including their tendency to degrade over time and that many do not adhere to a conserved structure. In this work, we describe a homology-based approach for the automatic identification of high-quality consensus TEs, aimed for use in the analysis of newly sequenced genomes.     

A comprehensive expression analysis of all members of a gene family encoding cell-wall enzymes allowed us to predict cis-regulatory regions involved in cell-wall construction in specific organs of Arabidopsis.

The Arabidopsis thaliana genome sequencing project has revealed that multigene families, such as those generated by genome duplications, are more abundant among plant genomes than among animal genomes. To gain insight into the evolutionary implications of the multigene families in higher plants, we examined the XTH gene family, a group of genes encoding xyloglucan endotransglucosylase/hydrolase, which are responsible for cell-wall construction in plants. Expression analysis of all members (33 genes) of this family, using quantitative real-time RT-PCR, revealed that most members exhibit distinct expression profiles in terms of tissue specificity and responses to hormonal signals, with some members exhibiting similar expression patterns. By comparing the flanking sequences of individual genes, we identified four sets of large-segment duplications and two sets of solitary gene duplications. In each set of gene duplicates, long nucleotide sequences, ranging from one to two hundred base pairs, are conserved. Furthermore, gene duplicates exhibit similar organ-specific expression profiles. These facts allowed us to predict putative cis-regulatory regions, particularly those responsible for cell-wall construction, and hence for morphogenesis, that are specific for certain organs or tissues in plants.     

Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes

Orf virus (OV) is an epitheliotropic poxvirus and belongs to the genus Parapoxvirus (PPV). PPV, especially OV, is regarded as a promising candidate for an expression vector. Among available live vaccines only strain D1701 represents a highly attenuated OV strain with clearly reduced pathogenicity. Therefore, we started to identify potentially non-essential genes or regions of D1701, which might be suitable for insertion and expression of foreign genes. The present contribution reviews some of the progress using the vegf-e (homologue of the mammalian vascular endothelial growth factor) gene locus for the generation of recombinant D1701. The vegf-e gene of D1701 is dispensable for virus growth in vitro and in vivo, and represents a major virulence determinant of OV. It is shown that foreign genes can be inserted and functionally expressed in the vegf-e locus, also leading to the induction of a specific immune response in the non-permissive host. Furthermore, it is reported that adaptation to VERO cells led to the deletion of three further regions of the OV D1701 genome, which seems to be combined with additional virus attenuation in sheep. Molecular analysis of this OV D1701 variant allows the identification of new, potentially non-essential sites in the viral genome.     

LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.     

最小基因组研究进展

随着许多生物体全基因组测序的完成,兴起了最小基因组的研究,即一个能营独立生活的生物体最少需要多少个基因。已知最小细胞支原体基因组是研究最小基因组的重要内容,还通过比较多种已测序基因组COG分析最小基因组,目前通过转座子插入基因突主为和同源重组删除基因的分析,进行最小基因组研究。     

Methods in comparative genomics: genome correspondence, gene identification and regulatory motif discovery.

In Kellis et al. (2003), we reported the genome sequences of S. paradoxus, S. mikatae, and S. bayanus and compared these three yeast species to their close relative, S. cerevisiae. Genomewide comparative analysis allowed the identification of functionally important sequences, both coding and noncoding. In this companion paper we describe the mathematical and algorithmic results underpinning the analysis of these genomes. (1) We present methods for the automatic determination of genome correspondence. The algorithms enabled the automatic identification of orthologs for more than 90% of genes and intergenic regions across the four species despite the large number of duplicated genes in the yeast genome. The remaining ambiguities in the gene correspondence revealed recent gene family expansions in regions of rapid genomic change. (2) We present methods for the identification of protein-coding genes based on their patterns of nucleotide conservation across related species. We observed the pressure to conserve the reading frame of functional proteins and developed a test for gene identification with high sensitivity and specificity. We used this test to revisit the genome of S. cerevisiae, reducing the overall gene count by 500 genes (10% of previously annotated genes) and refining the gene structure of hundreds of genes. (3) We present novel methods for the systematic de novo identification of regulatory motifs. The methods do not rely on previous knowledge of gene function and in that way differ from the current literature on computational motif discovery. Based on genomewide conservation patterns of known motifs, we developed three conservation criteria that we used to discover novel motifs. We used an enumeration approach to select strongly conserved motif cores, which we extended and collapsed into a small number of candidate regulatory motifs. These include most previously known regulatory motifs as well as several noteworthy novel motifs. The majority of discovered motifs are enriched in functionally related genes, allowing us to infer a candidate function for novel motifs. Our results demonstrate the power of comparative genomics to further our understanding of any species. Our methods are validated by the extensive experimental knowledge in yeast and will be invaluable in the study of complex genomes like that of the human.     

Genome-wide identification of NBS-encoding resistance genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.     

Gene identification in novel eukaryotic genomes by self-training algorithm

Finding new protein-coding genes is one of the most important goals of eukaryotic genome sequencing projects. However, genomic organization of novel eukaryotic genomes is diverse and ab initio gene finding tools tuned up for previously studied species are rarely suitable for efficacious gene hunting in DNA sequences of a new genome. Gene identification methods based on cDNA and expressed sequence tag (EST) mapping to genomic DNA or those using alignments to closely related genomes rely either on existence of abundant cDNA and EST data and/or availability on reference genomes. Conventional statistical ab initio methods require large training sets of validated genes for estimating gene model parameters. In practice, neither one of these types of data may be available in sufficient amount until rather late stages of the novel genome sequencing. Nevertheless, we have shown that gene finding in eukaryotic genomes could be carried out in parallel with statistical models estimation directly from yet anonymous genomic DNA. The suggested method of parallelization of gene prediction with the model parameters estimation follows the path of the iterative Viterbi training. Rounds of genomic sequence labeling into coding and non-coding regions are followed by the rounds of model parameters estimation. Several dynamically changing restrictions on the possible range of model parameters are added to filter out fluctuations in the initial steps of the algorithm that could redirect the iteration process away from the biologically relevant point in parameter space. Tests on well-studied eukaryotic genomes have shown that the new method performs comparably or better than conventional methods where the supervised model training precedes the gene prediction step. Several novel genomes have been analyzed and biologically interesting findings are discussed. Thus, a self-training algorithm that had been assumed feasible only for prokaryotic genomes has now been developed for ab initio eukaryotic gene identification.     

Patterns of evolutionary conservation of essential genes correlate with their compensability

Essential genes code for fundamental cellular functions required for the viability of an organism. For this reason, essential genes are often highly conserved across organisms. However, this is not always the case: orthologues of genes that are essential in one organism are sometimes not essential in other organisms or are absent from their genomes. This suggests that, in the course of evolution, essential genes can be rendered nonessential. How can a gene become non-essential? Here we used genetic manipulation to deplete the products of 26 different essential genes in Escherichia coli. This depletion results in a lethal phenotype, which could often be rescued by the overexpression of a non-homologous, non-essential gene, most likely through replacement of the essential function. We also show that, in a smaller number of cases, the essential genes can be fully deleted from the genome, suggesting that complete functional replacement is possible. Finally, we show that essential genes whose function can be replaced in the laboratory are more likely to be non-essential or not present in other taxa. These results are consistent with the notion that patterns of evolutionary conservation of essential genes are influenced by their compensability—that is, by how easily they can be functionally replaced, for example through increased expression of other genes.     

Detection of genomic islands via segmental genome heterogeneity

While the recognition of genomic islands can be a powerful mechanism for identifying genes that distinguish related bacteria, few methods have been developed to identify them specifically. Rather, identification of islands often begins with cataloging individual genes likely to have been recently introduced into the genome; regions with many putative alien genes are then examined for other features suggestive of recent acquisition of a large genomic region. When few phylogenetic relatives are available, the identification of alien genes relies on their atypical features relative to the bulk of the genes in the genome. The weakness of these ‘bottom–up’ approaches lies in the difficulty in identifying robustly those genes which are atypical, or phylogenetically restricted, due to recent foreign ancestry. Herein, we apply an alternative ‘top–down’ approach where bacterial genomes are recursively divided into progressively smaller regions, each with uniform composition. In this way, large chromosomal regions with atypical features are identified with high confidence due to the simultaneous analysis of multiple genes. This approach is based on a generalized divergence measure to quantify the compositional difference between segments in a hypothesis-testing framework. We tested the proposed genome island prediction algorithm on both artificial chimeric genomes and genuine bacterial genomes.     

一种基于代谢网络分析最小化基因组的方法及其在大肠杆菌中的应用

最小生命体的合成是合成生物学研究的重要方向。最小化基因组的同时而又不对细胞生长产生影响是代谢工程研究的一个重要目标。文中提出了一种从基因组尺度代谢网络模型出发,通过零通量反应删除及对非必需基因组合删除计算获得基因组最小化代谢网络模型的方法,利用该方法简化了大肠杆菌经典代谢网络模型iAF1260,由起始的1 260个基因简化得到了312个基因,而最优生物质生成速率保持不变。基因组最小化代谢网络模型预测了在细胞正常生长的前提下包含最少基因的代谢途径,为大肠杆菌获得最小基因组的湿实验设计提供了重要参考。     

A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae.

Autophagy is a starvation-induced transport pathway delivering parts of the cytosol into the lysosome (vacuole) for degradation. Autophagy significantly differs from other transport pathways by using double membrane layered transport intermediates. Based on the identification of autophagy genes in Saccharomyces cerevisiae, which served as a pacemaker for higher cells, our mechanistic knowledge of autophagy notably increased over the past few years. We here identify AUT8 as a novel gene essential for autophagy by screening a collection of approximately 5000 yeast deletion strains, each containing a defined deletion in an individual gene. This collection is a result of the world-wide Saccharomyces deletion project and covers the non-essential genes of the whole yeast genome. Homozygous aut8 Delta cells are impaired in maturation of proaminopeptidase I, and they fail to undergo the cell differentiation process of sporulation. The essential function of AUT8 for autophagy is further demonstrated by the lack of accumulation of autophagic vesicles in the vacuoles of aut8 Delta cells starved of nitrogen in the presence of the proteinase B inhibitor phenylmethylsulfonyl fluoride.     

Tests for gene clustering.

Comparing chromosomal gene order in two or more related species is an important approach to studying the forces that guide genome organization and evolution. Linked clusters of similar genes found in related genomes are often used to support arguments of evolutionary relatedness or functional selection. However, as the gene order and the gene complement of sister genomes diverge progressively due to large scale rearrangements, horizontal gene transfer, gene duplication and gene loss, it becomes increasingly difficult to determine whether observed similarities in local genomic structure are indeed remnants of common ancestral gene order, or are merely coincidences. A rigorous comparative genomics requires principled methods for distinguishing chance commonalities, within or between genomes, from genuine historical or functional relationships. In this paper, we construct tests for significant groupings against null hypotheses of random gene order, taking incomplete clusters, multiple genomes, and gene families into account. We consider both the significance of individual clusters of prespecified genes and the overall degree of clustering in whole genomes.     

Thermostability/infectivity defect caused by deletion of the core protein V gene in human adenovirus type 5 is rescued by thermo-selectable mutations in the core protein X precursor

Mastadenoviruses represent one of the four major genera of the Adenoviridae family comprising a variety of mammalian pathogens including human adenovirus (Ad), whose genomes encode a gene for minor core protein V (pV), not found in other genera of Adenoviridae. Deletion of other genus-specific genes (gene IX and E3 genes) from the Ad type 5 (Ad5) genome has been studied experimentally in vitro and the results on biological characterization of the mutants support the phylogenetic evidence of those genes being non-essential for Ad viability. On this basis it seemed logical to suggest that a deletion of gene V from the Ad5 genome could also be tolerated. To test this hypothesis we constructed and rescued the first pV-deletion mutant of human Ad5. As compared to Ad5, this mutant formed small plaques, had dramatically reduced thermostability and lower infectivity. A subsequent thermoselection screen of the pV-deleted Ad5 allowed isolation of a suppressor mutant Ad5-dV/TSB with restored biological characteristics. Since replication and viral assembly of Ad5-dV/TSB could still occur in the absence of pV, we conclude that pV is a non-essential component of the virion. The observed rescue of the biological defects appears to be associated with a cluster of point mutations in the gene encoding the precursor for the other core protein, X/Mu. This finding, thus, suggests possible roles of pV and protein X/Mu precursor in viral assembly. It also provides an interesting insight into genetic events that mediate molecular adaptation of viruses to possible changes in the genetic background in the course of their evolutionary divergence. The possible mechanism of the observed genetic suppression is discussed.     

Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution

In plant species with large genomes such as wheat or barley, genome organization at the level of DNA sequence is largely unknown. The largest sequences that are publicly accessible so far from Triticeae genomes are two 60 kb and 66 kb intervals from barley. Here, we report on the analysis of a 211 kb contiguous DNA sequence from diploid wheat (Triticum monococcum L.). Five putative genes were identified, two of which show similarity to disease resistance genes. Three of the five genes are clustered in a 31 kb gene-enriched island while the two others are separated from the cluster and from each other by large stretches of repetitive DNA. About 70% of the contig is comprised of several classes of transposable elements. Ten different types of retrotransposons were identified, most of them forming a pattern of nested insertions similar to those found in maize and barley. Evidence was found for major deletion, insertion and duplication events within the analysed region, suggesting multiple mechanisms of genome evolution in addition to retrotransposon amplification. Seven types of foldback transposons, an element class previously not described for wheat genomes, were characterized. One such element was found to be closely associated with genes in several Triticeae species and may therefore be of use for the identification of gene-rich regions in these species.     

虱目裂化线粒体基因组研究进展

虱目是哺乳类和鸟类体表的专性寄生虫。在虱科、阴虱科、长角鸟虱科和兽羽虱科的某些寄生虱种中发现了线粒体基因组裂化现象, 其线粒体基因组裂化成了多个环状的线粒体染色体, 如体虱(Pediculus humanus)、头虱(pediculus capitis)和阴虱(Pthirus pubis)的线粒体基因组分别裂化形成20个、20个和14个微环染色体。微环染色体可能是基因删除和同源重组的结果, 关于线粒体基因组裂化的具体原因和机制, 目前并不清楚, 推测可能是进化选择或随机遗传漂变的结果或与线粒体单链DNA结合蛋白的缺失有关。鉴于线粒体基因组裂化研究对于深入理解线粒体的起源和进化方面具有重要意义, 文章以虱目裂化线粒体基因组为主线, 列举了动物裂化线粒体基因组和裂化特征, 阐述了虱目裂化线粒体基因组的研究现状, 分析了虱目线粒体基因组裂化的类型、原因和机制, 并对该领域未来的研究方向进行了展望。     

Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.