大肠杆菌最小基因组分析和删减进展

大肠杆菌是基础研究最透彻、应用广泛的微生物,构建含减小甚至是最小基因组的大肠杆菌将为合成生物学的研究和应用提供理想的底盘生物。介绍了大肠杆菌最小基因组的生长与繁殖必需基因的生物信息学分析和实验鉴定,基因组敲除技术,以及删减基因组的大肠杆菌菌株的构建和应用等方面的研究进展。     

The revolution of the biology of the genome

Sequence data of entire eukaryotic genomes and their detailed comparison have provided new evidence on genome evolution. The major mechanisms involved in the increase of genome sizes are polyploidization and gene duplication.Subsequent gene silencing or mutations, preferentially in regulatory sequences of genes, modify the genome and permit the development of genes with new properties. Mechanisms such as lateral gene transfer, exon shuffling or the creation of new genes by transposition contribute to the evolution of a genome, but remain of relatively restricted relevance.Mechanisms to decrease genome sizes and, in particular, to remove specific DNA sequences, such as blocks of satellite DNAs, appear to involve the action of RNA interference (RNAi). RNAi mechanisms have been proven to be involved in chromatin packaging related with gene inactivation as well as in DNA excision during the macronucleus development in ciliates.     

Rate asymmetry after genome duplication causes substantial long-branch attraction artifacts in the phylogeny of Saccharomyces species

Whole-genome duplication (WGD) produces sets of gene pairs that are all of the same age. We therefore expect that phylogenetic trees that relate these pairs to their orthologs in other species should show a single consistent topology. However, a previous study of gene pairs formed by WGD in the yeast Saccharomyces cerevisiae found conflicting topologies among neighbor-joining (NJ) trees drawn from different loci and suggested that this conflict was the result of "asynchronous functional divergence" of duplicated genes (Langkjaer, R. B., P. F. Cliften, M. Johnston, and J. Piskur. 2003. Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. Nature 421:848-852). Here, we test whether the conflicting topologies might instead be due to asymmetrical rates of evolution leading to long-branch attraction (LBA) artifacts in phylogenetic trees. We constructed trees for 433 pairs of WGD paralogs in S. cerevisiae with their single orthologs in Saccharomyces kluyveri and Candida albicans. We find a strong correlation between the asymmetry of evolutionary rates of a pair of S. cerevisiae paralogs and the topology of the tree inferred for that pair. Saccharomyces cerevisiae gene pairs with approximately equal rates of evolution tend to give phylogenies in which the WGD postdates the speciation between S. cerevisiae and S. kluyveri (B-trees), whereas trees drawn from gene pairs with asymmetrical rates tend to show WGD pre-dating this speciation (A-trees). Gene order data from throughout the genome indicate that the "A-trees" are artifacts, even though more than 50% of gene pairs are inferred to have this topology when the NJ method as implemented in ClustalW (i.e., with Poisson correction of distances) is used to construct the trees. This LBA artifact can be ameliorated, but not eliminated, by using gamma-corrected distances or by using maximum likelihood trees with robustness estimated by the Shimodaira-Hasegawa test. Tests for adaptive evolution indicated that positive selection might be the cause of rate asymmetry in a substantial fraction (19%) of the paralog pairs.     

Correlated asymmetry of sequence and functional divergence between duplicate proteins of Saccharomyces cerevisiae

The role of sequence divergence in functional divergence of duplicate genes is a topic of great interest. In this study, we compare the numbers of amino acid substitutions in each sequence since two yeast duplicates diverged, using a preduplication ancestral outgroup. Using this strategy, we explored the relationship between sequence divergence and functional divergence between duplicate partners. We show that the degree of relative functional asymmetry between duplicate proteins is proportional to the relative sequence divergence between them. Furthermore, of the two duplicates, the copy closer to their ancestral sequence (fewer number of amino acid substitutions) interacts with more proteins and affects fitness more severely when deleted. Therefore, asymmetric sequence divergence between duplicates is correlated with asymmetric functional divergence and may underlie the duplicate's role in genetic robustness against mutations. Among the functional traits considered, protein abundance appears to have the strongest correlation with the nonsynonymous divergence between duplicates. Taken together with the results from whole-genome analyses, our results indicate that within-species duplicates are subject to the same evolutionary force that acts on interspecific sequence and functional divergence. In particular, we detect signs of purifying selection on the more slowly evolving duplicate.     

Detection and Characterization of Fungal-Specific Proteins in Saccharomyces cerevisiae

In this study, I searched for fungal-specific proteins in the genome of the budding yeast Saccharomyces cerevisiae, inferred from a comparison of amino acid sequences. I used the GTOP (Genomes to Protein structures and functions) database of the DDBJ (DNA Data Bank of Japan), which consists of 21 genomes from Archaea, 203 genomes from Bacteria, and 50 genomes from Eucarya (including 18 fungal genomes). Among 5,874 proteins of S. cerevisiae, 1,551 have homologs only in Eucarya, and 504 of the 1,551 have homologs only in fungi. To find fungal-specific proteins, homologs of the homologs have been searched repeatedly. As a result, 132 of the 504 are characterized as fungal-specific proteins. The genes encoding the 132 fungal-specific proteins are not included in the list of essential genes for viability in the S. cerevisiae genome deletion project. Among the 132 proteins, 99 are S. cerevisiae-specific, and no protein that is distributed among 10 or more of the 18 fungal species exists. In addition, most of the fungal-specific proteins are very small and functionally unknown. My results show that the fungal-specific proteins have short evolutionary histories, suggesting that S. cerevisiae produces novel proteins and that ancestral fungi also produced small proteins most of which have disappeared or have been combined with other proteins during fungal evolution.     

Prediction of nucleosome positions in the yeast genome based on matched mirror position filtering

Nucleosome positioning can affect the accessibility of the underlying DNA to the nuclear environment and as such plays an essential role in the regulation of cellular processes. Specific patterns have been found in the underlying DNA sequences of the nucleosome, and one of the most important patterns includes dinucleotides distributed every 10 to 11 base pairs. Based on this property, we propose to match each dinucleotide in the sequence against its mirror occurrences for 10 to 11 base pairs on both left-hand and right­hand sides. A large number of matches in a local region will then signify the existence of a nucleosome. In this paper, we propose the matched mirror position filters for efficient matching of periodic dinucleotide patterns and computationally predict the nucleosome positions. Experimental results on the Saccharomyces cerevisiae (yeast) genome show that the proposed algorithm can predict nucleosome positions effectively. More than 50% of our predicted nucleosomes are within 35 base pairs of those detected by biological experiments.     

Major transitions in evolution by genome fusions: from prokaryotes to eukaryotes,metazoans, bilaterians and vertebrates

Journal of Structural and Functional Genomics - The major transitions in human evolution from prokaryotes toeukaryotes, from protozoans to metazoans, from the first animals tobilaterians and...     

The evolutionary demography of duplicate genes

Although gene duplication has generally been viewed as a necessary source of material for the origin of evolutionary novelties, the rates of origin, loss, and preservation of gene duplicates are not well understood. Applying steady-state demographic techniques to the age distributions of duplicate genes censused in seven completely sequenced genomes, we estimate the average rate of duplication of a eukaryotic gene to be on the order of 0.01/gene/million years, which is of the same order of magnitude as the mutation rate per nucleotide site. However, the average half-life of duplicate genes is relatively small, on the order of 4.0 million years. Significant interspecific variation in these rates appears to be responsible for differences in species-specific genome sizes that arise as a consequence of a quasi-equilibrium birth-death process. Most duplicated genes experience a brief period of relaxed selection early in their history and a minority exhibit the signature of directional selection, but those that survive more than a few million years eventually experience strong purifying selection. Thus, although most theoretical work on the gene-duplication process has focused on issues related to adaptive evolution, the origin of a new function appears to be a very rare fate for a duplicate gene. A more significant role of the duplication process may be the generation of microchromosomal rearrangements through reciprocal silencing of alternative copies, which can lead to the passive origin of post-zygotic reproductive barriers in descendant lineages of incipient species.     

2R or not 2R: Testing hypotheses of genome duplication in early vertebrates

The widely popular hypothesis that there were two rounds of genome duplication by polyploidization early in vertebrate history (the 2R hypothesis) has been difficult to test until recently. Among the lines of evidence adduced in support of this hypothesis are relative genome size, relative gene number, and the existence of genomic regions putatively duplicated during polyploidization. The availability of sequence for a substantial portion of the human genome makes possible the first rigorous tests of this hypothesis. Comparison of gene family size in the human genome and in invertebrate genomes shows no evidence of a 4:1 ratio between vertebrates and invertebrates. Furthermore, explicit phylogenetic tests for the topology expected from two rounds of polyploidization have revealed alternative topologies in a substantial majority of human gene families. Likewise, phylogenetic analyses have shown that putatively duplicated genomic regions often include genes duplicated at widely different times over the evolution of life. The 2R hypothesis thus can be decisively rejected. Rather, current evidence favors a model of genome evolution in which tandem duplication, whether of genomic segments or of individual genes, predominates.     

Gradual genome stabilisation by progressive reduction of the Saccharomyces uvarum genome in an interspecific hybrid with Saccharomyces cerevisiae

Considerable amounts of molecular and genetic data indicate that interspecific hybridisation may not be rare among natural strains of Saccharomyces sensu stricto. Although a post-zygotic barrier operating during meiosis usually prevents the production of viable spores, stable hybrids can arise which can even evolve into distinct species. This study was aimed to analyse the genome of a fertile Saccharomyces cerevisiae x S. uvarum hybrid and monitor its changes over four filial generations of viable spores. The molecular genetic analysis demonstrated that the two species did not contribute equally to the formation and stabilisation of the hybrid genome. S. cerevisiae provided the mitochondrial DNA and the more stable part of the nuclear genome. The S. uvarum part of the hybrid nuclear genome became progressively smaller by loosing complete chromosomes and genetic markers in the course of successive meiotic divisions. Certain S. uvarum chromosomes were eliminated and/or underwent rearrangements in interactions with S. cerevisiae chromosomes. Numerous S. uvarum chromosomes acquired S. cerevisiae telomere sequences. The gradual elimination of large parts of the S. uvarum genome was associated with a progressive increase of sporulation efficiency. We hypothesise that this sort of genomic alterations may contribute to speciation in Saccharomyces sensu stricto.     

Whole genome duplication of intra- and inter-chromosomes in the tomato genome

Whole genome duplication(WGD)events have been proven to occur in the evolutionary history of most angiosperms.Tomato is considered a model species of the Solanaceae family.In this study,we describe the details of the evolutionary process of the tomato genome by detecting collinearity blocks and dating the WGD events on the tree of life by combining two different methods:synonymous substitution rates(Ks)and phylogenetic trees.In total,593 collinearity blocks were discovered out of 12 pseudo-chromosomes constructed. It was evident that chromosome 2 had experienced an intra-chromosomal duplication event.Major inter-chromosomal duplication occurred among all the pseudo-chromosome.We calculated the Ks value of these collinearity blocks.Two peaks of Ks distribution were found,corresponding to two WGD events occurring approximately 36-82 million years ago(MYA)and 148-205 MYA.Additionally, the results of phylogenetic trees suggested that the more recent WGD event may have occurred after the divergence of the rosidasterid clade,but before the major diversification in Solanaceae.The older WGD event was shown to have occurred before the divergence of the rosid-asterid clade and after the divergence of rice-Arabidopsis(monocot-dicot).     

The complete chloroplast genome sequence of Pelargonium x hortorum: organization and evolution of the largest and most highly rearranged chloroplast genome of land plants

The chloroplast genome of Pelargonium x hortorum has been completely sequenced. It maps as a circular molecule of 217,942 bp and is both the largest and most rearranged land plant chloroplast genome yet sequenced. It features 2 copies of a greatly expanded inverted repeat (IR) of 75,741 bp each and, consequently, diminished single-copy regions of 59,710 and 6,750 bp. Despite the increase in size and complexity of the genome, the gene content is similar to that of other angiosperms, with the exceptions of a large number of pseudogenes, the recognition of 2 open reading frames (ORF56 and ORF42) in the trnA intron with similarities to previously identified mitochondrial products (ACRS and pvs-trnA), the losses of accD and trnT-ggu and, in particular, the presence of a highly divergent set of rpoA-like ORFs rather than a single, easily recognized gene for rpoA. The 3-fold expansion of the IR (relative to most angiosperms) accounts for most of the size increase of the genome, but an additional 10% of the size increase is related to the large number of repeats found. The Pelargonium genome contains 35 times as many 31 bp or larger repeats than the unrearranged genome of Spinacia. Most of these repeats occur near the rearrangement hotspots, and 2 different associations of repeats are localized in these regions. These associations are characterized by full or partial duplications of several genes, most of which appear to be nonfunctional copies or pseudogenes. These duplications may also be linked to the disruption of at least 1 but possibly 2 or 3 operons. We propose simple models that account for the major rearrangements with a minimum of 8 IR boundary changes and 12 inversions in addition to several insertions of duplicated sequence.     

The age of the Arabidopsis thaliana genome duplication

We estimate the timing of the Arabidopsis thaliana whole-genome duplication by means of phylogenetic and statistical analysis, and propose two possible scenarios for the duplication. The first one, based on the assumption that the duplicated segments diverged from an autotetraploid form, places the duplication at about 38 million years ago, after the Arabidopsislineage diverged from that of soybean (Glycine max) and before it diverged from its sister genus, Brassica. The second scenario assumes that the ancestor was allotetraploid, and suggests that the duplication is younger than 38 million years and may have contributed to the Arabidopsis-Brassica divergence. In each case, our estimate places the age of the genome duplication as significantly younger than previously reported.     

Komagataeibacter rhaeticus3-15全基因组及葡萄糖脱氢酶基因缺失体的构建

细菌纤维素(BC)是一种新型的可再生、可降解的生物高分子材料.为了最大程度的发挥BC生产菌株K.rhaeticus 315的生产能力,本文首先对K.rhaeticus 315进行全基因组测序,通过功能基因的注释、分析碳源代谢流向.结果 显示,该菌株碳代谢特征之一是缺乏磷酸果糖激酶的编码基因,不能通过EMP途径代谢糖类碳...     

In silico evidence for functional specialization after genome duplication in yeast

A fairly recent whole-genome duplication (WGD) event in yeast enables the effects of gene duplication and subsequent functional divergence to be characterized. We examined 15 ohnolog pairs (i.e. paralogs from a WGD) out of c . 500 Saccharomyces cerevisiae ohnolog pairs that have persisted over an estimated 100 million years of evolution. These 15 pairs were chosen for their high levels of asymmetry, i.e. within the pair, one ohnolog had evolved much faster than the other. Sequence comparisons of the 15 pairs revealed that the faster evolving duplicated genes typically appear to have experienced partially – but not fully – relaxed negative selection as evidenced by an average nonsynonymous/synonymous substitution ratio (d N /d S _avg=0.44) that is higher than the slow-evolving genes' ratio (d N /d S _avg=0.14) but still <1. Increased number of insertions and deletions in the fast-evolving genes also indicated loosened structural constraints. Sequence and structural comparisons indicated that a subset of these pairs had significant differences in their catalytically important residues and active or cofactor-binding sites. A literature survey revealed that several of the fast-evolving genes have gained a specialized function. Our results indicate that subfunctionalization and even neofunctionalization has occurred along with degenerative evolution, in which unneeded functions were destroyed by mutations.