Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.     

问号赖型钩端螺旋体的进化分析

 对问号赖型钩端螺旋体的复制基因进行了分析,引入以S为分歧时间的概念,通过对每个同义位点和非同义位点的核苷酸替代数目的比较,并利用最小二乘法进行了函数拟合,得出在早期复制基因受到相对松的净化选择,之后选择约束力不断增强并慢慢趋于稳定.     

钩端螺旋体LAg42膜蛋白基因的克隆及原核表达

分别以问号状赖型钩体017株，56601株及双曲钩体PatocI株基因组为模板，PCR扩增目的基因lag42与质粒pET32a(＋)构建重组原核表达质粒，在大肠杆菌BL21中诱导表达.结果显示不同毒力赖型钩体均能扩增出约1100 bp的片段，而PatocI株则未能扩增出目的片段；不同赖型钩体的lag42基因的同源性很高.融合表达质粒pET-lag42转化大肠杆菌BL21后，经IPTG诱导，表达出约62kDa的重组融合蛋白，用Western blotting证实其有良好的抗原性，纯化蛋白免疫新西兰大白兔，获     

江西地区鼠源致病性钩端螺旋体菌的分离纯化与鉴定

目的对江西地区钩端螺旋体菌进行分离纯化与鉴定,为后期建立江西地区钩端螺旋体菌的发病模型提供菌源。方法通过捕捉钩端螺旋体疫源地的储存宿主——褐家鼠,无菌操作摘取肾脏,将肾脏接种到EMJH培养基中培养、纯化菌株。对纯化后的钩端螺旋体用钩体外膜脂蛋白LipL32基因引物进行PCR检测以鉴定菌株,并对分离菌对金黄地鼠的致病性进行了研究。结果分离纯化到一株钩端螺旋体菌,PCR扩增为阳性,经核苷酸序列测定分析证实上述序列与钩端螺旋体菌FDAARGOS 2 03基因序列（Gen Bank登录号:CP020414. 2）同源性为100%。分离菌对金黄地鼠具有一定的致病性。结论我们成功从江西地区储存宿主褐家鼠肾脏中分离到一株具有致病性的钩端螺旋体菌株。本次研究为以后分离纯化钩端螺旋体菌累积了宝贵的经验,通过收集江西省内流行的钩端螺旋体菌型,为钩端螺旋体菌的发病模型提供菌源,并以此为基础,研发出可靠的临床早期诊断产品,以期达到有效控制江西省地区钩端螺旋体病的目的,降低公共卫生隐患。     

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.     

基于酵母基因组高通量数据的基因-基因相互作用预测

后基因组时代的显著特点是大规模基因组和蛋白质组实验平台所产生的大量高通量数据,整合并利用基因组和蛋白组信息成为这一时代的主要挑战之一. 因此,基因-基因相互作用将有助于理解细胞内基因之间的相互作用以及信号传导通路研究提供有价值的参考. 为预测酵母基因组中基因-基因相互作用,我们利用高通量数据中的蛋白-蛋白相互作用、遗传表型数据、基因微阵列表达数据以及功能基因注释数据等来分析酵母中的基因-基因相互作用. 本文建立的预测方法为在系统水平上理解酵母基因组中的基因功能提供了依据,也为揭示酵母基因组中的基因-基因相互作用网络奠定理论基础.     

小鼠睾丸特异表达新基因(mtLR1)在大肠杆菌中的表达

根据已经克隆的mtLR1基因序列,采用RT-PCR的方法获得mtLR1基因。将所得的PCR产物插入原核表达载体pMAL-P-2x中,得重组质粒(pMAL-P-2x/mtLR1)并转化大肠杆菌(E．coli)DH5α．经IPTG诱导表达,SDS-PAGE电泳分析显示,重组蛋白得到了正确表达,表达的融合蛋白占菌体蛋白总量的52％,分子质量约为73 kDa。mtLR1蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础。     

A novel domain-based method for predicting the functional classes of proteins

Prediction of protein functions from known genomic sequences is an important mission of bioinformatics. One approach is to classify proteins into functional categories. We have therefore developed a method based on protein domain composition and the maximum likelihood estimation (MLE) algorithm to classify proteins according to functions. Using the Saccharomyces cerevisiae genome, we compared the effectiveness of the MLE approach with that of an intuitive and simple method. The MLE method outperformed the simple method, achieving an estimated specificity of 75.45 % and an estimated sensitivity of 40.26 %. These results indicate that domain is an important feature of proteins and is closely related to protein function.     

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana

The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans--the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.     

Multifunctions of histone H1 proteins

Among various histones, histone H1 proteins have been appreciated for their multiple functions in diverse biological processes. In addition to being a structural protein in chromatin, H1 proteins also play critical roles in cell cycle, gene expression, and development. Recent studies reveal the possible effects of H1 in some diseases, such as cancer and neurodegenerative diseases. Here, we review different variants of HI, the functions, and post translational modifications of ill variants are also discussed.     

Transmembrane structure and function of dctPQM encoding C4－dicarboxylate transport proteins from nitrogen－fixing P. stutzeri A1501

C4-dicarboxylate transport proteins of diazotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ,and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene encoding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii.Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively.The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ2L The resultant plasmid was named pSZY6.By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501,randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was constructed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20,10 and 5mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A150L This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.     

衣藻的核基因组转化及外源基因转录分析

通过珠磨法将衣藻表达载体pSP108转入到莱茵衣藻细胞壁缺陷型CC-400藻株中,经抗性筛选及PCR鉴定,获得了24个转化藻株.在抗性基因ble编码序列的中间部位和末端部位分别设计两对不同的探针引物,运用实时荧光定量PCR对转基因藻株进行外源基因转录水平的分析.结果发现:不同转化藻株外源基因的转录水平有明显差异;同一转化藻株两对探针引物,外源基因的转录水平也存在差异.说明莱茵衣藻在转化过程中,外源基因整合到基因组上的片段数量及片段长度和完整性具有不确定性.     

桃钙调磷酸酶B类似蛋白互作蛋白激酶基因家族鉴定与分析

钙调磷酸酶B类似蛋白互作蛋白激酶(CIPKs)在植物生长发育和抗逆过程中发挥着重要作用。为了对桃中CIPK家族基因进行系统分析,利用桃基因组数据库,通过生物信息学手段,鉴定桃CIPK家族基因的基因结构、染色体定位和编码蛋白,通过序列比对进行进化和分类分析。结果表明,桃基因组中含有18个CIPK基因,分布于桃的6条染色体上。MEME和Pfam保守结构域分析显示,桃CIPK蛋白均含有2个保守的PKinase和NAF结构域。进化树分析表明CIPKs可分为2个亚家族。Net Phos 2.0 Server结果显示Pp CIPKs存在着大量的丝氨酸(Ser)、苏氨酸(Thr)及酪氨酸(Tyr)潜在磷酸化位点。以上结果将为今后揭示桃CIPK蛋白的功能提供重要的理论基础。     

白色念珠菌基因组研究进展

目的:介绍白色念珠菌基因组的最新研究进展.方法:查阅国内外大量关于白色念珠菌基因组研究的文献并进行综合详述.结果:白色念珠菌是第一个完成全基因组测序的致病性真核生物,在基因组序列信息研究上取得了重大进展,在基因功能、转录组、蛋白质组等方面获得了许多重要成果.结论:白色念珠菌的基因组研究成果将对研究白色念珠菌的致病机制、耐药机制以及抗真菌药物开发有重大的意义.     

小链霉菌HCCB10043敲除硫酯酶基因的工程菌株构建及其代谢产物的研究

小链霉菌(Streptomyces parvus)HCCB10043的主要代谢产物为脂肽类化合物A21978C,其基因组序列中包括了非核糖体肽合成酶(NRPS non ribosomal peptide synthetase)、聚酮合酶(PKS polyketide synthases)以及NRPS-PKS混合的多酶体系基因簇,它们的共同特点是在代谢产物生物合成簇中连接有一个硫酯酶域,即TE(thioesterase)domain.硫酯酶可以使已经合成的化合物链的合成过程终止,并且具有水解释放成熟脂肽以及环化线性脂肽链的功能.通过对联二吡啶类代谢产物合成簇中TE基因的敲除构得工程菌株,工程菌发酵结果表明联二吡啶类代谢产物产量减少.     

Blast fungus-induction and developmental and tissue-specific expression of a rice P450<Emphasis Type="Italic">CYP72A</Emphasis> gene cluster

Cytochrome P450 gene superfamily is widely involved in diverse processes of plant development and environmental responses including defense response to pathogens.We previously isolated a rice cDNA fragment in a DD-PCR screening for blast fungus-induced genes. In the current study, we isolated a CYP72A gene cluster consisting of 7 P450 CYP72A genes (CYP72A17-23) with the conserved cDNA sequence through the public rice genome data. There are total 14 putative CYP72A members in the rice genome, with high diversity at N-terminal sequences while high homology at C-terminal sequences of those 14 putative proteins. We analyzed expression profiles of the cloned 7 CYP72A genes during pathogen infection and development. The results showed that expression of CYP72A18, 19, 22 and 23 was differentially regulated in the incompatible and compatible interactions between rice and blast fungus. Except CYP72A20, a pseudogene, other 6 CYP72A genes also exhibited temporal and spatial expression patterns, respectively.These findings provide fundamental data for rice P450 gene function analysis.     

阿特拉津降解菌Enterobacter sp.的基因差异分析

筛选并分析生物降解菌Enterobacter sp.在阿特拉津作用下的差异基因。以Enterobacter sp.BIDMC 29基因组为参考,利用高通量测序技术,对阿特拉津降解菌株和对照菌株进行转录组测序分析。结果表明,共有368个基因表现出显著差异,其中上调基因231个,下调基因137个。在差异基因GO富集分析的前30个条目中,尿嘧啶分解代谢、氮利用、硝酸盐同化、硫化氢生物合成、裂解酶活性和过氧化物酶活性等相关基因表现富集。Pathway分析显示,差异基因涉及70条代谢途径,与氮代谢、氨基酸合成和代谢、ABC转运蛋白、丙酮酸代谢等过程有关。菌株通过调节基因表达来提高对阿特拉津的降解能力,为进一步解析阿特拉津的代谢机制奠定基础。     

Genome survey uncovers the secrets of sex and lifestyle in caterpillar fungus

The caterpillar fungus Ophiocordyceps sinensis (best known as Cordyceps sinensis) mummifies ghost moth larvae exclusively in Tibetan Plateau alpine ecosystems. Touted as "Himalayan Viagra", the fungus is highly prized due to its medical benefits and dwindling supplies. Attempts to culture the sexual fruiting-body have failed and the huge market demand has led to severe devastation of local ecosystems and to the fungus heading towards extinction. By genome sequencing, we establish that unlike related insect pathogens O. sinensis contains two compatible mating-type genes in its genome and is self-fertile, i.e. homothallic. However, sexual processes are only initiated under native environmental conditions. O. sinensis resembles biotrophic plant pathogens in having a genome shaped by retrotransposon-driven expansions. The resulting changes in gene content suggest that O. sinensis has a biphasic pathogenic mechanism beginning with stealth pathogenesis in early host instars. O. sinensis is the first psychrophilic fungus sequenced and is adapted to extreme cold with putative antifreeze proteins and mechanisms for increasing lipid accumulation and fatty acid unsaturation. We hypothesize that for the inbreeding O. sinensis the massive proliferation of retrotransposons provides a tradeoff between the advantages of increased genetic variation independent of sexual recombination and deletion of genes dispensable for its specialized pathogenic lifestyle.     

Transgene directionally integrated into C-genome of Brassica napus

Transgenic Brassica napus has been widely planted in Canada, the United States, and some other countries. In China, although the policy for genetically modified foods has not yet opened, genetically modified rape- seed oil as raw material for biodiesel of…