Proteome-scale analysis of biochemical activity

During the last 10 years, there has been a large increase in the number of genome sequences available for study, altering the way that the biology of organisms is studied. In particular, scientific attention has increasingly focused on the proteome, and specifically on the role of all the proteins encoded by the genome. We focus here on several aspects of this problem. We describe several technologies in widespread use to clone genes on a genome-wide scale, and to express and purify the proteins encoded by these genes. We also describe a number of methods that have been developed to analyze various biochemical properties of the proteins, with attention to the methodology and the limitations of the approaches, followed by a look at possible developments in the next decade.     

植物内生链霉菌Streptomyces sp. SAT1的基因组测序和比较基因组分析

【背景】一直以来,链霉菌都是活性物质的主要生产者,近年来随着抗生素滥用引起的环境和微生物抗药性问题越发严重,挖掘高效生物防治因子和新型抗生素成为了解决以上问题的重要手段。【目的】通过获得植物内生链霉菌SAT1全基因组序列和次级代谢基因簇信息,利用比较基因组学和泛基因组学分析SAT1菌株的特殊性以及与其他链霉菌的共性,为阐明SAT1抑菌和内生机制提供理论基础,为揭示链霉菌的生态功能提供可靠数据。【方法】通过三代测序平台PacBio Sequel完成SAT1基因组测序,利用生物信息学技术进行注释和功能基因分类;分别利用RAxML和PGAP软件进行系统发育树的构建和泛基因组分析;次级代谢基因簇的预测和分析通过antiSMASH网站完成。【结果】获得SAT1菌株的全基因组完成图,该菌线性染色体长度约7.47 Mb,包含有4个质粒,GC含量近73%,共预测到7 550个蛋白编码基因,含有37个次级代谢基因簇,分属29个类型,其中默诺霉素基因簇与加纳链霉菌具有较高相似性。42株代表链霉菌中,单个菌株次级代谢基因簇数量为20-55个,主要类型为PKS类、Terpene类和Nrps类,而且含有大量杂合基因簇,各个菌株中特有基因数目较为庞大。【结论】链霉菌SAT1菌株在基因组特点以及次级代谢基因簇的数量和类型上与其余41株链霉菌具有一定的共性,其中潮霉素B基因簇和默诺霉素基因簇合成的相关物质可能与SAT1抑菌活性密切相关。42株链霉菌中次级代谢基因簇数量的多少与基因组大小成正相关,同时大量杂合基因簇以及庞大的特有基因数目的存在说明链霉菌在长期进化过程中存在了很高程度的水平基因转移现象,可能具有重要的生态功能。     

Organization of the mitochondrial genome of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> (anamorphic Hypocreales)

A 33.4-kb fragment of the mitochondrial genome of Fusarium oxysporum has been sequenced. The fragment contains the complete gene sequences for 13 of the 14 proteins typically encoded by the mitochondrial genome of filamentous ascomycetes. Similarity searching revealed all encoded proteins to be most similar to those from other members of the Hypocreales. The fragment contains the complete small subunit rRNA gene, partial large rRNA subunit gene, and 12 tRNAs. Two introns were present, one in the nad5 gene and one in the large rRNA subunit gene, the latter containing a ribosomal protein gene.     

Protein sequences of linked genes are highly conserved in two bacterial species

It has been shown that proteins encoded by linked genes have similar rates of evolution and that clusters of essential genes are found in regions with low recombination rates. We show here that proteins encoded by linked genes in two closely related bacterial species, namely Escherichia coli K12 and Salmonella typhimurium LT2, evolve more slowly when compared with proteins encoded by genes that are not linked as assessed by protein sequence similarity. The proteins encoded by the identified linked genes share an average sequence identity of 82.5% compared with a 46.5% identity of proteins encoded by genes that are not linked.     

Evolutionary analyses of non‐family genes in plants

There are a large number of ‘non‐family’ (NF) genes that do not cluster into families with three or more members per genome. While gene families have been extensively studied, a systematic analysis of NF genes has not been reported. We performed comparative studies on NF genes in 14 plant species. Based on the clustering of protein sequences, we identified ~94 000 NF genes across these species that were divided into five evolutionary groups: Viridiplantae wide, angiosperm specific, monocot specific, dicot specific, and those that were species specific. Our analysis revealed that the NF genes resulted largely from less frequent gene duplications and/or a higher rate of gene loss after segmental duplication relative to genes in both low‐copy‐number families (LF; 3–10 copies per genome) and high‐copy‐number families (HF; >10 copies). Furthermore, we identified functions enriched in the NF gene set as compared with the HF genes. We found that NF genes were involved in essential biological processes shared by all plant lineages (e.g. photosynthesis and translation), as well as gene regulation and stress responses associated with phylogenetic diversification. In particular, our analysis of an Arabidopsis protein–protein interaction network revealed that hub proteins with the top 10% most connections were over‐represented in the NF set relative to the HF set. This research highlights the roles that NF genes may play in evolutionary and functional genomics research.     

鲍曼不动杆菌携带前噬菌体的生物信息学分析

[目的] 预测并分析82株全基因组测序的鲍曼不动杆菌前噬菌体的携带情况，鉴定前噬菌体编码的抗生素耐药基因和毒力因子。[方法] 利用PHASTER （Phage Search Tool Enhanced Release）软件预测鲍曼不动杆菌携带的前噬菌体，采用CARD （The Comprehensive Antibiotic Research Database）和VFDB （Virulence Factors Database）在线分析软件预测前噬菌体编码的抗生素耐药基因和毒力因子。[结果] 预测到472条鲍曼不动杆菌前噬菌体，其中完整型前噬菌体201条，疑似型前噬菌体91条，缺陷型前噬菌体180条。平均每株鲍曼不动杆菌基因组中可携带至少2条完整型前噬菌体。每株鲍曼不动杆菌所携带的全部前噬菌体占其基因组比例约为4%-6%。29条前噬菌体携带77个耐药基因，耐药表型共有14种，分别来自15个不同的家族，涵盖6种抗生素耐药的作用机制。132条前噬菌体编码毒力基因，归类为38种毒力基因和34种毒力因子。不同类型的前噬菌体普遍携带1-2种毒力因子，少数前噬菌体携带3种及以上毒力因子。分析毒力因子可能的宿主来源构成比发现，除鲍曼不动杆菌外，脑膜炎奈瑟菌、痢疾志贺氏菌、嗜肺军团菌及其亚种等也有较高的结构比例，是可能的宿主来源。[结论] 鲍曼不动杆菌普遍携带前噬菌体，但前噬菌体基因在鲍曼不动杆菌基因组中所占比例不高。部分前噬菌体携带抗生素耐药基因，以氨基糖苷类、磺胺类及β-内酰胺类耐药为主。约30%的前噬菌体携带毒力基因。前噬菌体可能在鲍曼不动杆菌抗生素耐药性的获得、传播及致病性演变中发挥重要作用。     

枯草芽孢杆菌N2-10的基因组测序和比较基因组分析

【背景】枯草芽孢杆菌N2-10是一株具有较强抑菌能力且能产纤维素酶等多种水解酶的革兰氏阳性菌,在发酵饲料中具有较大的应用潜力。【目的】通过获得枯草芽孢杆菌N2-10的全基因组序列信息,进一步解析菌株次级代谢产物合成基因信息,并通过比较基因组学分析菌株N2-10与模式菌株的差异性,为阐明N2-10抑菌和益生机制提供理论基础。【方法】通过二代Illumina NovaSeq联合三代PacBio Sequel测序平台,对菌株N2-10进行全基因组测序,将测序数据进行基因组组装、基因预测与功能注释,并利用比较基因组学分析N2-10与其他菌株的差异。【结果】菌株N2-10基因组大小为4 036 899 bp,GC含量为43.88%;共编码4 163个编码基因,所有编码基因总长度为3594369bp,编码区总长度占基因组总长度的89.04%;含有85个tRNA、10个5S rRNA、10个16S rRNA、10个23S rRNA,以及2个CRISPR-Cas、1个前噬菌体和6个基因岛;在GO (gene ontolog)、COG (clusters of orthologous groups of...     

Molecular genetics of nucleotide sugar interconversion pathways in plants

Nucleotide sugar interconversion pathways represent a series of enzymatic reactions by which plants synthesize activated monosaccharides for the incorporation into cell wall material. Although biochemical aspects of these metabolic pathways are reasonably well understood, the identification and characterization of genes encoding nucleotide sugar interconversion enzymes is still in its infancy. Arabidopsis mutants defective in the activation and interconversion of specific monosaccharides have recently become available, and several genes in these pathways have been cloned and characterized. The sequence determination of the entire Arabidopsis genome offers a unique opportunity to identify candidate genes encoding nucleotide sugar interconversion enzymes via sequence comparisons to bacterial homologues. An evaluation of the Arabidopsis databases suggests that the majority of these enzymes are encoded by small gene families, and that most of these coding regions are transcribed. Although most of the putative proteins are predicted to be soluble, others contain N-terminal extensions encompassing a transmembrane domain. This suggests that some nucleotide sugar interconversion enzymes are targeted to an endomembrane system, such as the Golgi apparatus, where they may co-localize with glycosyltransferases in cell wall synthesis. The functions of the predicted coding regions can most likely be established via reverse genetic approaches and the expression of proteins in heterologous systems. The genetic characterization of nucleotide sugar interconversion enzymes has the potential to understand the regulation of these complex metabolic pathways and to permit the modification of cell wall material by changing the availability of monosaccharide precursors.     

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

Background

The number of species with completed genomes, including those with evidence for recent whole genome duplication events has exploded. The recently sequenced Atlantic salmon genome has been through two rounds of whole genome duplication since the divergence of teleost fish from the lineage that led to amniotes. This quadrupoling of the number of potential genes has led to complex patterns of retention and loss among gene families.

Results

Methods have been developed to characterize the interplay of duplicate gene retention processes across both whole genome duplication events and additional smaller scale duplication events. Further, gene expression divergence data has become available as well for Atlantic salmon and the closely related, pre-whole genome duplication pike and methods to describe expression divergence are also presented. These methods for the characterization of duplicate gene retention and gene expression divergence that have been applied to salmon are described.

Conclusions

With the growth in available genomic and functional data, the opportunities to extract functional inference from large scale duplicates using comparative methods have expanded dramatically. Recently developed methods that further this inference for duplicated genes have been described.

     

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, β-sheet motifs and stretches of α-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.     

Genome analysis and genetic enhancement of tomato

The Solanaceae is an important family of vegetable crops, ornamentals and medicinal plants. Tomato has served as a model member of this family largely because of its enriched cytogenetic, genetic, as well as physical, maps. Mapping has helped in cloning several genes of importance such as Pto, responsible for resistance against bacterial speck disease, Mi-1.2 for resistance against nematodes, and fw2.2 QTL for fruit weight. A high-throughput genome-sequencing program has been initiated by an international consortium of 10 countries. Since heterochromatin has been found to be concentrated near centromeres, the consortium is focusing on sequencing only the gene-rich euchromatic region. Genomes of the members of Solanaceae show a significant degree of synteny, suggesting that the tomato genome sequence would help in the cloning of genes for important traits from other Solanaceae members as well. ESTs from a large number of cDNA libraries have been sequenced, and microarray chips, in conjunction with wide array of ripening mutants, have contributed immensely to the understanding of the fruit-ripening phenomenon. Work on the analysis of the tomato proteome has also been initiated. Transgenic tomato plants with improved abiotic stress tolerance, disease resistance and insect resistance, have been developed. Attempts have also been made to develop tomato as a bioreactor for various pharmaceutical proteins. However, control of fruit quality and ripening remains an active and challenging area of research. Such efforts should pave the way to improve not only tomato, but also other solanaceous crops.     

一株类志贺邻单胞菌噬菌体生物学特性及全基因组分析

[目的]多重耐药菌株的出现给食品安全带来严重威胁.噬菌体是不同于抗生素的一类重要杀菌因子,对其生物学特性及基因组的研究和分析可为噬菌体的抗菌应用提供依据.[方法]对噬菌体phiP4-7的生物学特性、基因组学、分类学进行研究.[结果]经透射电子显微镜观察,确定phiP4-7头部直径为(50.59±1.68) nm,尾部长...     

ASTRO-FOLD: a combinatorial and global optimization framework for Ab initio prediction of three-dimensional structures of proteins from the amino acid sequence

The field of computational biology has been revolutionized by recent advances in genomics. The completion of a number of genome projects, including that of the human genome, has paved the way toward a variety of challenges and opportunities in bioinformatics and biological systems engineering. One of the first challenges has been the determination of the structures of proteins encoded by the individual genes. This problem, which represents the progression from sequence to structure (genomics to structural genomics), has been widely known as the structure-prediction-in-protein-folding problem. We present the development and application of ASTRO-FOLD, a novel and complete approach for the ab initio prediction of protein structures given only the amino acid sequences of the proteins. The approach exhibits many novel components and the merits of its application are examined for a suite of protein systems, including a number of targets from several critical-assessment-of-structure-prediction experiments.     

Improved genomic resources and new bioinformatic workflow for the carcinogenic parasite Clonorchis sinensis: Biotechnological implications

Clonorchis sinensis (family Opisthorchiidae) is an important foodborne parasite that has a major socioeconomic impact on ~35 million people predominantly in China, Vietnam, Korea and the Russian Far East. In humans, infection with C. sinensis causes clonorchiasis, a complex hepatobiliary disease that can induce cholangiocarcinoma (CCA), a malignant cancer of the bile ducts. Central to understanding the epidemiology of this disease is knowledge of genetic variation within and among populations of this parasite. Although most published molecular studies seem to suggest that C. sinensis represents a single species, evidence of karyotypic variation within C. sinensis and cryptic species within a related opisthorchiid fluke (Opisthorchis viverrini) emphasise the importance of studying and comparing the genes and genomes of geographically distinct isolates of C. sinensis. Recently, we sequenced, assembled and characterised a draft nuclear genome of a C. sinensis isolate from Korea and compared it with a published draft genome of a Chinese isolate of this species using a bioinformatic workflow established for comparing draft genome assemblies and their gene annotations. We identified that 50.6% and 51.3% of the Korean and Chinese C. sinensis genomic scaffolds were syntenic, respectively. Within aligned syntenic blocks, the genomes had a high level of nucleotide identity (99.1%) and encoded 15 variable proteins likely to be involved in diverse biological processes. Here, we review current technical challenges of using draft genome assemblies to undertake comparative genomic analyses to quantify genetic variation between isolates of the same species. Using a workflow that overcomes these challenges, we report on a high-quality draft genome for C. sinensis from Korea and comparative genomic analyses, as a basis for future investigations of the genetic structures of C. sinensis populations, and discuss the biotechnological implications of these explorations.     

The Comparative Genomics of Polyglutamine Repeats: Extreme Difference in the Codon Organization of Repeat-Encoding Regions Between Mammals and Drosophila

Polyglutamine repeats within proteins are common in eukaryotes and are associated with neurological diseases in humans. Many are encoded by tandem repeats of the codon CAG that are likely to mutate primarily by replication slippage. However, a recent study in the yeast Saccharomyces cerevisiae has indicated that many others are encoded by mixtures of CAG and CAA which are less likely to undergo slippage. Here we attempt to estimate the proportions of polyglutamine repeats encoded by slippage-prone structures in species currently the subject of genome sequencing projects. We find a general excess over random expectation of polyglutamine repeats encoded by tandem repeats of codons. We nevertheless find many repeats encoded by nontandem codon structures. Mammals and Drosophila display extreme opposite patterns. Drosophila contains many proteins with polyglutamine tracts but these are generally encoded by interrupted structures. These structures may have been selected to be resistant to slippage. In contrast, mammals (humans and mice) have a high proportion of proteins in which repeats are encoded by tandem codon structures. In humans, these include most of the triplet expansion disease genes. Received: 17 August 2000 / Accepted: 20 November 2000     

不同来源植物乳杆菌基因组结构和功能差异的比较研究

[目的] 本试验研究不同来源植物乳杆菌（Lactobacillus plantarum）基因特点以及在不同环境下其基因多样性,探究2株L.plantarum A8和P9在肠道生境及植物表面适应性的异同,为优良菌株的开发提供理论基础。[方法] 本研究对从动物肠道和植物表面分离获得的L.plantarum A8和L.plantarum P9的基因组进行分析,利用第二代测序技术（NextGeneration Sequencing,NGS）,基于Illumina NovaSeq测序平台,同时利用第三代单分子测序技术,基于PacBio Sequel测序平台,对L.plantarum A8和L.plantarum P9进行测序。采用Carbohydrate-active enzymes（CAZy）、Koyto encyclopedia of genes and genomes（KEGG）和Clusters of orthologous genes（COG）数据库对基因组进行功能注释;采用CGView软件绘制菌株的基因组环形图谱。应用比较基因组学与已经公开发表的其他L.plantarum基因组进行比较分析。[结果] 由研究可知L.plantarum A8和L.plantarum P9基因组大小存在差异,通过构建系统发育树发现2株菌与其他来源的L.plantarum分在同一分支,并且L.plantarum P9与母乳来源的L.plantarum WLPL04菌株距离最近,而L.plantarum A8与L.paraplantarum DSM10667距离最近。通过基因家族分析可知,2株菌共有基因为2643个,其中包括一些抗应激蛋白如热休克蛋白、冷休克蛋白。L.plantarum A8和P9独特基因分别为321和336个,L.plantarum A8中独特基因主要参与DNA复制、ABC转运系统（ABC transfer system）、PTS系统（phosphotransferase system）、磺酸盐转运系统、氨基酸生物合成等代谢通路;L.plantarum P9的独特基因以参与碳水化合物的运输和代谢基因居多,例如rpiA基因、lacZ基因、FruA基因等。[结论] 通过比较基因组学方法解析L.plantarum的基因组信息,发现动物肠道来源的L.plantarum具有较好的氨基酸转运能力,植物表面附着的L.plantarum菌株具有较好碳水化合物利用能力,从而为益生菌的开发与利用提供理论依据。     

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana

We analyzed the Arabidopsis thaliana genome sequence to detect Late Embryogenesis Abundant (LEA) protein genes, using as reference sequences proteins related to LEAs previously described in cotton or which present similar characteristics. We selected 50 genes representing nine groups. Most of the encoded predicted proteins are small and contain repeated domains that are often specific to a unique LEA group. Comparison of these domains indicates that proteins with classical group 5 motifs are related to group 3 proteins and also gives information on the possible history of these repetitions. Chromosomal gene locations reveal that several LEA genes result from whole genome duplications (WGD) and that 14 are organized in direct tandem repeats. Expression of 45 of these genes was tested in different plant organs, as well as in response to ABA and in mutants (such as abi3, abi5, lec2 and fus3) altered in their response to ABA or in seed maturation. The results demonstrate that several so-called LEA genes are expressed in vegetative tissues in the absence of any abiotic stress, that LEA genes from the same group do not present identical expression profile and, finally, that regulation of LEA genes with apparently similar expression patterns does not systematically involve the same regulatory pathway.