细菌外毒素序列中特有模体的识别及其基因本体注释分析

【目的】识别细菌外毒素序列中特有模体,进一步理解外毒素的致病机制。【方法】构建非致病性细菌蛋白质数据库,利用InterProScan对数据库中非致病菌蛋白质序列以及收集的经实验确认的89条细菌外毒素蛋白质序列进行模体搜索。【结果】在89条细菌外毒素序列中,分析得到了39个细菌外毒素特有模体。【结论】得到的外毒素特有模体与外毒素功能密切相关,为在致病性细菌基因组内搜索外毒素序列奠定了基础;同时通过对外毒素特有模体的基因本体(Gene ontology,GO)注释分析,进一步阐明了细菌外毒素的致病机制。     

稻瘟菌Magnaporthe oryzae P-ATPases基因家族分析

利用TCDB(Transporter Classification Database)网站数据库中的P-ATPases氨基酸序列对稻瘟菌全基因组表达序列(Coding Sequence,CDS)数据库进行搜索和分析,共发现23个P-ATPases基因,进化树分析表明这23个基因分属于4个家族和7个亚家族。构建了本地ESTs数据库,通过P-ATPases基因CDS序列与EST序列比对分析发现,在这些基因中有20个存在EST同源体,另外3个基因没有发现EST同源体,因此这20个基因是真实P-ATPases基因的可能性更高。运用MEME程序分析了这些P-ATPases蛋白结构域的基序,有6种基序在90%以上的基因氨基酸序列中出现,属保守基序。对这23个P-ATPases基因GC含量的分析表明,它们的平均GC含量在0.519-0.628之间,稍高于稻瘟菌整个基因组GC平均含量(0.516),同时这些基因内各区段GC含量变化不大,没有明显的梯度变化。本文结果为下一步深入研究稻瘟菌中P-ATPases基因家族的功能奠定了基础。     

噬病毒体启动子模体的初步预测分析

噬病毒体作为遗传信息的承载者和传播者,在生态系统中有着举足轻重的作用。为了探索噬病毒体以及噬病毒体与其宿主巨病毒间的调控模式和进化关系,本文基于MEME启动子预测分析工具,对噬病毒体ORF上游部分序列(包括起始密码子ATG)中的启动子模体进行了预测分析。依据预测得到的启动子模体E值和在基因组上的位置、数量和长度等信息,共得到17个潜在的启动子模体。sputnik的模体2和zamilon的模体2都存在一个富含AT的区域,广泛分布在基因组上,可能调控大部分ORF的表达;mavirus启动子模体中存在TCTA盒子,而ALM启动子模体中存在ATCT盒子,这两个模体可能为后期启动子模体;OLV的模体2、YSLV1和2的模体3、YSLV3的模体1以及YSLV4的模体1和2都存在富含AT的区域,广泛存在各基因组上,为各噬病毒体的潜在启动子模体。该结果为进一步研究噬病毒体ORF的表达时序及其与宿主巨病毒之间的协同调控表达奠定了一定的理论基础。     

致病菌III型分泌系统分子伴侣序列保守性分析及新伴侣的预测

很多革兰氏阴性致病菌使用Ⅲ型分泌系统(type Ⅲ secretion system,TTSS)将毒力因子直接注射到宿主细胞质中,其中某些效应蛋白(即毒素)需要专一的Ⅲ型分泌系统分子伴侣才能有效分泌。尽管2000年Cheng等提出这些分子伴侣中应该存在保守的氨基酸序列或保守的分泌信号,但是以往的研究并没有发现保守的氨基酸序列或分泌信号。文章作者通过生物信息学模体搜索对45个Ⅲ型分泌系统分子伴侣的氨基酸序列进行分析,找出5个与Ⅲ型分泌系统分子伴侣功能有关的保守模体和模体组合。其中一个为已知的,一个比已知的34氨基酸多肽重复(tetratdco peptide repeat,TPR)模体更具有Ⅲ型分泌系统分子伴侣SycD家族特征,其他3个为新的模体。每个Ⅲ型分泌系统分子伴侣家族包含一个或多个模体,这揭示了同类分子伴侣序列中确实存在保守的位点,暗示着同类分子伴侣可能存在相同的空间结构和功能,进一步支持了Birtanlan有关相同的空间结构具有普遍TTSS三维分泌信号的假说,并基于此分析和同源分析预测出27个新的可能的Ⅲ型分泌系统分子伴侣。     

水稻锚蛋白基因家族分析和锚定膜蛋白的表达模式研究

锚蛋白重复序列模体是生物体内最普遍的蛋白质序列模体之一,在多种细胞活动中主要介导蛋白质与蛋白质的相互作用。采用生物信息学的方法,在基因组水平上搜索和鉴定了水稻锚定重复序列蛋白,通过分析蛋白质二级结构,进行水稻锚蛋白基因家族的聚类分析,并在此基础上,用RT-PCR的方法分析水稻锚定膜蛋白的表达模式,为水稻锚蛋白基因的研究提供了理论依据。     

链球菌属纤连蛋白结合蛋白的生物信息学分析

对链球菌属纤连蛋白结合蛋白进行系统的分析,找出其特征,为以后的研究奠定基础。通过MUSCLE、BLASTP、PSI-BLAST、PHYLIP、Perl编程等生物信息学方法对217条候选纤连蛋白结合蛋白进行研究。发现共有31个序列纤连蛋白结合蛋白重复单元序列模体,绝大部分蛋白由两个或两个以上的不同模体序列构成,其保守结构域组合呈现多样性等特征。表明链球菌属纤连蛋白结合蛋白重复单元序列模体不是严格统一的,总体上具有变异性,但模体之间又有很大的相似性。     

细胞黏附分子互作网络的构建与分析

借助网络分析可对基因调控、蛋白质互作和信号转导等细胞活动进行全局和局部性质分析.以细胞黏附的蛋白质相互作用为对象,通过数据挖掘和可视化软件构建了整合蛋白介导的黏附分子互作网络,该分子互作网络由156种蛋白质通过690种相互作用相连,其平均节点度为8.66、平均聚集系数为0.24,平均路径长度为2.6.黏附分子互作网络中包含数个功能模块,这些模块涉及网络内部多种分子相互作用的启动与停止,并进一步影响细胞的黏附、迁移和骨架组织.对黏附分子网络进行模体筛选和比较,发现一些数量相对较少、以三元复合物为主要结构的关键模体,同时对各网络模块和模体对细胞黏附的调控作用进行了探讨.     

DNA保守序列识别算法的并行化和MPI集群环境构建

DNA序列中保守序列的识别需要较大的计算量。开发了一个转录因子结合位点识别的并行算法,能够从多条DNA序列中识别指定长度的序列模式。算法使用概率模型进行序列模式保守性的度量,利用迭代过程实现保守序列的搜索。使用C编程结合MPI消息传递模型开发了相应的程序,并在Windows平台下构建了一个3节点的集群环境,利用20个长度均为200的序列数据集进行测试,实现了模体识别工作,结果表明并行算法使模体识别的效率得到提高。     

基于对数线性模型的酵母基因转录调控模体分析

识别真核基因的转录因子结合位点(或称模体)是后基因组时代的一项主要工作,对共表达或共调控的基因同时进行分析可以提高模体识别的准确性.本文基于2×2列联表的对数线性模型,以模体出现的基因条数计数,对酵母核糖体蛋白(RP)基因普遍使用的转录调控模体进行分析,然后用U-检验进一步筛选出相对于背景序列来说过表达的模体.这些模体为酵母RP基因潜在的转录调控元件,与实验获得的转录因子结合位点的符合率达90%.本方法的优点在于用严格的统计标准在一组基因启动子中搜索普遍使用的模体,克服了以往分析中对模体使用普遍性的模糊判断.本文的方法也可以有效地搜索共表达基因族的组合调控模体对.研究中还发现一个现象:2×2列联表中反映属性相关程度的Pearson相关系数与对数线性模型的交互效应之间存在着明显的相关性.这一结果提示,可以用对数线性模型的交互效应来评价两属性的关联情况.     

人类1号染色体DNA序列8-mer的相对模体数分布及8-mer使用的进化分离

以人类1号染色体DNA序列为样本,分别计算了CDS、5'UTR、3'UTR、内含子和基因间五类序列中8-mer出现的频数,并得到8-mer相对模体数随频数的分布。发现内含子和基因间序列是明显的三峰分布,CDS是单峰分布,5'UTR和3'UTR是近似单峰分布。为了揭示这些分布出现差异的原因,将8-mer集合按照8-mer中包含两个或两个以上某二核苷酸(XY2)、包含一个某二核苷酸(XY1)和包含0个某二核苷酸(XY0)进行分类。发现16种分类中,只有CGj分类的三个子集分别形成独立的单峰分布。表明DNA序列是由三类CGj子集组成的,它们出现的频数是独立进化的结果;实际的8-mer频数分布是这三个CGj频数分布的叠加;由于这三个分布的距离不同,才造成了五类序列中8-mer分布的差异。对五类序列CGj三个子集中二核苷酸和三核苷酸出现的相对频数进行分析,发现CG2模体的相对频数在五类序列中基本相同,CG1模体的相对频数可将五类序列明显区分,CG0模体的相对频数可将编码序列和非编码序列明显区分。总之,CGj模体集合在DNA序列的组成上具有特定的规律性,在DNA序列进化上扮演了重要的角色。     

细菌萜类合成酶的生物信息学分析

【目的】目前对于萜类合成酶(Terpenoid synthase,TPS)的研究主要集中在植物和真菌中,而对细菌TPS的系统研究尚少。建立在大量已经被测序的细菌基因组基础上,利用生物信息学方法,对细菌TPS在全基因组范围内进行识别、分类和功能分析。【方法】利用TPS的隐马尔科夫模型(Pfam编号为PF03936)搜索自建的细菌蛋白质组数据库,预测出细菌TPS。对这些候选TPS的蛋白序列用MAFFT 7.130b进行多序列比对,并利用MEGA 6.0对多序列比对结果进行进化分析。利用MEME和PredictProtein分别进行细菌TPS的基序(Motifs)和点突变分析。【结果】建立在生物信息学分析的基础上,1 423条细菌TPS被识别,它们分布在8个门中,即放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)、酸杆菌门(Acidobacteria)和衣原体门(Chlamydiae)。进化分析表明细菌TPS可分为4大类,Motifs分析表明除了各类之间保守的基序(Motifs)外,还有特异的Motifs,这暗示着细菌TPS在不同类别之间的功能分化。点突变分析表明,细菌TPS不同位点的氨基酸突变对TPS功能的影响不同。【结论】细菌TPS主要分布于8个门中,其中在2个门中细菌TPS尚未见报道,即厚壁菌门(Firmicutes)与酸杆菌门(Acidobacteria)。基于进化分析,可以把细菌TPS分为4类,各类之间的差异可能是由类特异的Motifs决定的,另外细菌TPS不同氨基酸位点的突变分析为今后验证TPS的功能提供了很好的理论基础。     

Comparative sequence analysis of imprinted genes between human and mouse to reveal imprinting signatures

We performed a comparative genomic sequence analysis between human and mouse for 24 imprinted genes on human chromosomes 1, 6, 7, 11, 13, 14, 15, 18, 19, and 20. The MEME program was used to search for motifs within conserved sequences among the imprinted genes and we then used the MAST program to analyze for the presence or absence of motifs in the imprinted genes and 128 nonimprinted genes. Our analysis identified 15 motifs that were significantly enriched in the imprinted genes. We generated a logistic regression model by combining multiple motifs as input variables and the 24 imprinted genes and the 128 nonimprinted genes as a training set. The accuracy, sensitivity, and specificity of our model were 98, 92, and 99%, respectively. The model was further validated by an open test on 12 additional imprinted genes. The motifs identified in this study are novel imprinting signatures, which should improve our understanding of genomic imprinting and the role of genomic imprinting in human diseases.     

Greedy mixture learning for multiple motif discovery in biological sequences

MOTIVATION: This paper studies the problem of discovering subsequences, known as motifs, that are common to a given collection of related biosequences, by proposing a greedy algorithm for learning a mixture of motifs model through likelihood maximization. The approach adds sequentially a new motif to a mixture model by performing a combined scheme of global and local search for appropriately initializing its parameters. In addition, a hierarchical partitioning scheme based on kd-trees is presented for partitioning the input dataset in order to speed-up the global searching procedure. The proposed method compares favorably over the well-known MEME approach and treats successfully several drawbacks of MEME. RESULTS: Experimental results indicate that the algorithm is advantageous in identifying larger groups of motifs characteristic of biological families with significant conservation. In addition, it offers better diagnostic capabilities by building more powerful statistical motif-models with improved classification accuracy.     

Searching for protein signatures using a multilevel alphabet

Short motifs are known to play diverse roles in proteins, such as in mediating the interactions with other molecules, binding to membranes, or conducting a specific biological function. Standard approaches currently employed to detect short motifs in proteins search for enrichment of amino acid motifs considering mostly the sequence information. Here, we presented a new approach to search for common motifs (protein signatures) which share both physicochemical and structural properties, looking simultaneously at different features. Our method takes as an input an amino acid sequence and translates it to a new alphabet that reflects its intrinsic structural and chemical properties. Using the MEME search algorithm, we identified the proteins signatures within subsets of protein which encompass common sequence and structural information. We demonstrated that we can detect enriched structural motifs, such as the amphipathic helix, from large datasets of linear sequences, as well as predicting common structural properties (such as disorder, surface accessibility, or secondary structures) of known functional‐motifs. Finally, we applied the method to the yeast protein interactome and identified novel putative interacting motifs. We propose that our approach can be applied for de novo protein function prediction given either sequence or structural information. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

The Fusobacterium nucleatum outer membrane protein RadD is an arginine-inhibitable adhesin required for inter-species adherence and the structured architecture of multispecies biofilm

A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central 'bridging organisms' in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine-inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine-binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum . Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD ) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive 'early oral colonizers'. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation.     

Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis

The proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high-iron conditions. Fluorescence and immuno-cytochemical experiments show that high-iron-grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low-iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti-AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two-dimensional electrophoresis and immunoblot of adhesins from surface-labelled parasites provided evidence that all members of the multigene family were co-ordinately expressed and placed on the trichomonal surface. Similar two-dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface-expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug-resistant isolate absent in hydrogenosome proteins and adhesins, were non-adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms.     

Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence

The first step in the bacterial colonization and infection of uropathogenic Escherichia coli is adherence to uroepithelium. Over 80% of all urinary tract infections are caused by E. coli. Uropathogenic E. coli express several adherence factors including type 1 and P fimbriae, which mediate attachment to the uroepithelium through specific binding to different glycoconjugate receptors. We showed that P and type 1 fimbriae are not the sole adhesins on uropathogenic E. coli and sialic acid also mediates nonspecific bacterial adherence of uropathogenic E. coli and urinary bladder epithelium.     

A bioinformatic strategy for the detection, classification and analysis of bacterial autotransporters

Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.