适冷海洋细菌交替假单胞菌（Pseudoalteromonas sp.）MB-1内切葡聚糖酶基因的克隆和表达

从黄海深海海底淤泥中筛选出一株产纤维素酶的适冷革兰氏阴性杆菌MB1,克隆和分析了MB1的16S rDNA序列（GenBank接受号：AY551321）,经鉴定为交替假单胞菌（Pseudoalt eromonas）,命名为Pseudoalteromonas sp.MB1。克隆了该菌适冷内切葡聚糖酶基因celA（GenBank接受号：AY551322）,并在大肠杆菌（Escherichia coli）BL21中进行了表达。重组E.coli菌体破碎后,获取上清液,其中融合蛋白GSTCelA浓度约为78.5mg/L。分析了融合酶GSTCelA的性质,其最适反应温度为35℃,最适反应pH值为72,为中性适冷酶。实验结果为交替假单胞菌低温纤维素酶的基础理论和应用研究奠定了基础。     

一株多环芳烃降解菌的鉴定及GST基因克隆和序列分析

由石油污染土壤中分离到一株能以多环芳烃（菲、芴、萘）为唯一碳源的细菌,经形态观察、生理生化（BiologGN）和 G+C mol%分析,鉴定该菌为少动鞘氨醇单胞菌(Sphingomonas paucimobilis)。与16S rDNA序列同源性的比较进一步确证了鉴定结果。经菲诱导后的细菌谷胱甘肽S转移酶（Glutathione Stransferase, GST）酶活明显高于未诱导前,表明谷胱甘肽S转移酶可能与多环芳烃的降解有关。根据该酶基因的同源性序列设计引物,PCR扩增出编码谷胱甘肽S转移酶基因片段,进一步证实在该菌中有GST的存在。测序后基于编码GST的基因所进行的系统发育分析表明,该多环芳烃降解菌与其它多环芳烃降解菌在进化上亲缘关系较近。     

极端嗜热古菌——芝田硫化叶菌DNA解旋酶的分离纯化和性质研究

采用Qsepharose离子交换层析、磷酸纤维素P1 1吸附层析、肝素琼脂糖吸附层析、Su perdex 2 0 0凝胶过滤和PhenylSuperose疏水层析等步骤 ,从嗜酸热芝田硫化叶菌细胞裂解液中分离纯化了一个DNA解旋酶。该解旋酶具有受DNA激活的ATP酶活性。根据SDS PAGE测定结果 ,该酶的分子质量约为 63kD。芝田硫化叶菌DNA解旋酶可以解开底物上 70bp的双链区 ,其解旋活性依赖于双链区旁的单链分叉。该解旋酶的活性依赖于Mg2 + 和ATP的水解 ,在NaCl浓度超过 2 0 0mmol L时受到抑制。该酶的最适pH为 6 7。该酶在 40℃～ 80℃之间均有活性 ,70℃时活性最高。芝田硫化叶菌DNA解旋酶是从古菌中分离得到的第一个天然DNA解旋酶。     

耐盐及苯乙酸、甲基对硫磷降解基因工程菌的构建

H1（Halomonas sp.）是一株耐高盐浓度（18% NaCl, W/V）和降解苯乙酸的菌株,pDT3质粒为pUC19插入甲基对硫磷水解酶基因（mpd基因）构建而成。采用ＨindⅢ酶切,获得含有完整mpd基因片段,克隆到广宿主质粒pKT230和pBBR1MCS2上,构建成质粒pKTMP和pBBRMP。通过三亲杂交,在辅助质粒pRK2013的帮助下,将质粒pKTMP和pBBRMP转移到Ｈ１中,得到的工程菌HpKTMP和HpBBRMP具有耐盐、降解苯乙酸和水解甲基对硫磷的功能,其中HpBBRMP水解酶活性与亲本菌株甲基对硫磷降解菌（Pseudomonas putida）DLLE4相当,而HpKTMP水解酶活性要提高1倍左右。经过传代试验,证明了工程菌的稳定性。     

硫酸软骨素酶产生菌的筛选及酶的分离纯化

利用平板快速筛选法,从鱼腹中筛选到一株产生硫酸软骨素酶的细菌YH311,通过生物特性和生化反应试验考察,初步鉴定为温和气单胞菌（Aeromonas sobria）。培养至36h时,该菌株产酶达到高峰期,培养液的酶活力达09U/mL。利用超声波破碎菌体细胞,分别测定培养液和菌体细胞的酶活力,发现培养液的酶活力要远远大于菌体细胞的酶活力,显示该酶为分泌型表达,属于胞外酶。发酵液经离心后,上清液用硫酸铵分级沉淀,再分别经过CMCellulose、QAEsephadex A50和Sephadex G150层析柱进行逐级分纯,并跟踪酶活,最后获得硫酸软骨素酶。SDSPAGE检测为单一条带,其分子量约为80kD。     

两株产生免疫抑制剂的小双孢菌分离株的鉴定

从江苏无锡土壤中分离到两株玫瑰小双孢菌SIPI226和SIPI207,经形态、化学分析、Ribotyping及16S rRNA分析,两菌株细胞壁含meso\|DAP、磷酸类脂PIV、无枝菌酸,醌为MK9(H0,H2,H4),G+C mol%分别为683和694。经初步鉴定为玫瑰小双孢菌的两个新亚种：玫瑰小双孢菌无锡亚种(Microbispora rosea subsp. wuxiensis)和玫瑰小双孢菌鼋头渚亚种(Microbispora rosea subsp. yuantouzhuensis)。菌株SIPI226和SIPI207分别为玫瑰小双孢菌无锡亚种和玫瑰小双孢菌鼋头渚亚种的典型菌株。     

编码1,3-丙二醇氧化还原酶基因的克隆和表达

采用PCR法克隆了巴氏梭菌（Clostridium pasteurianum）CpN86菌株编码1,3丙二醇氧化还原酶基因（dhaT基因）;完成了dhaT基因测序、表达载体构建和在大肠杆菌中表达;分离和纯化了dhaT基因表达的重组蛋白。实验结果：（1）PCR法克隆的dhaT基因和肺炎克雷伯氏菌Klebsiella pneumoniae菌株dhaT基因的序列同源性为829%;（2）dhaT基因表达蛋白的酶活为108U/mg;（3）dhaT基因表达的蛋白分子量为43kD;（4）Western blot确定了dhaT基因表达的蛋白和 CpN86菌株天然蛋白有相同的抗原反应。     

芝田硫化叶菌Ssh7蛋白的进化保守性及DNA结合特性

嗜酸热古菌——芝田硫化叶菌(Sulfolobus shibatae)合成大量7 kD DNA结合蛋白Ssh7. Southern杂交结果显示, 该菌基因组中含有两个编码Ssh7蛋白的基因, 分别命名为ssh7a和ssh7b. 对这两个基因进行了克隆、序列测定和在大肠杆菌中的表达. 测序结果表明, 两个Ssh7多肽仅在3个氨基酸位置上有差异; 此外, ssh7a和ssh7b的顺式调控序列也十分相似, 提示存在着维持两个基因的序列和表达都不变的选择压力. 杂交结果还显示, 与芝田硫化叶菌一样, 硫磺矿硫化叶菌(Sulfolobus solfataricus)基因组中也含有两个编码7 kD蛋白的基因. 结合其他报道, 这一结果提示编码7 kD蛋白的基因可能在硫化叶菌种间分歧形成之前发生了基因复制. 采用电泳迁移率改变试验(EMSA)分析了天然及重组Ssh7蛋白与双链DNA片段之间的相互作用. 天然和重组蛋白的EMSA行为相似, 说明Ssh7与DNA的相互作用既不受发生在天然蛋白赖氨酸残基上的甲基化的影响, 也不受两种多肽异构体之间在序列上的差异的影响. 在所采用的实验条件下, Ssh7与双链DNA片段结合时的结合位点大约为6.6 bp, 表观解离常数为(0.7~1.0)×10^-7 mol/L. 此外, Ssh7与负超螺旋DNA的结合强于与线性和松弛DNA的结合.     

力复霉素产生菌地中海拟无枝菌酸菌U32丙氨酸脱氢酶基因克隆及表达

丙氨酸脱氢酶(EC1411)可逆催化丙氨酸脱氨生成丙酮酸和NADH。它是生物体内的氨基酸代谢和氨同化途径的关键酶。在地中海拟无枝菌酸菌(Amycolatopsis mediterranei)U32中,丙氨酸脱氢酶的活力与力复霉素的生物合成有负相关现象,其活力受KNO3全局效应的调控。根据结核分枝杆菌(Mycobacterium tuberculosis)和天蓝链霉菌(Streptomyces coelicolor)的丙氨酸脱氢酶氨基酸的保守序列和地中海拟无枝菌酸菌U32对氨基酸密码子的使用偏好,设计一对简并PCR引物。以此引物从地中海拟无枝菌酸菌U32中扩增到一555bp的片段,并以此片段为探针从地中海拟无枝菌酸菌U32 基因组cosmid文库中成功的克隆到了丙氨酸脱氢酶结构基因(ald)。它编码了一个371个氨基酸的蛋白质,基因的GC含量为72.5％,符合链霉菌的基因结构特征。在起始密码子的上游6个碱基处,有一典型的链霉菌核糖体结合位点(RBS)：AGGAGG,第75位的氨基酸为赖氨酸,是丙酮酸结合位点。以pET28b为载体,在E.coli BL21(DE3)中高效表达了ald基因。用IPTG在22℃时诱导得到的丙氨酸脱氢酶活力最高。用HisTag柱纯化了表达的丙氨酸脱氢酶。酶学性质研究表明该酶专一性以LAla和NAD(H)为底物。     

卡瑞苯西思伯克霍尔德氏菌卤乙酸脱卤酶的分离纯化及性质

从云南西北部土样中分离到一株卡瑞苯西思伯克霍尔德氏菌(Burkholderia caribensis),它在氯乙酸培养基中能产生较高活性的卤乙酸脱卤酶。经硫酸铵盐析、DEAE SephadexA50柱层析、羟基磷灰石柱层析、Sephadex G200 凝胶过滤后,获得电泳纯酶。用SDSPAGE测定酶分子量为46kD。水解氯乙酸的Km值为3.7×10-3mol/L。酶反应的最适温度为40℃,最适pH值为9.5。金属离子及CN-、EDTA对该酶有不同程度的影响,Hg2+和CN-则对该酶有强烈的抑制作用。     

Geminiviruses: a tale of a plasmid becoming a virus

Background  

Geminiviruses (family Geminiviridae) are small single-stranded (ss) DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps) of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons.     

Testing a Primary and a Secondary Endpoint in a Group Sequential Design

Summary We consider a clinical trial with a primary and a secondary endpoint where the secondary endpoint is tested only if the primary endpoint is significant. The trial uses a group sequential procedure with two stages. The familywise error rate (FWER) of falsely concluding significance on either endpoint is to be controlled at a nominal level α. The type I error rate for the primary endpoint is controlled by choosing any α‐level stopping boundary, e.g., the standard O'Brien–Fleming or the Pocock boundary. Given any particular α‐level boundary for the primary endpoint, we study the problem of determining the boundary for the secondary endpoint to control the FWER. We study this FWER analytically and numerically and find that it is maximized when the correlation coefficient ρ between the two endpoints equals 1. For the four combinations consisting of O'Brien–Fleming and Pocock boundaries for the primary and secondary endpoints, the critical constants required to control the FWER are computed for different values of ρ. An ad hoc boundary is proposed for the secondary endpoint to address a practical concern that may be at issue in some applications. Numerical studies indicate that the O'Brien–Fleming boundary for the primary endpoint and the Pocock boundary for the secondary endpoint generally gives the best primary as well as secondary power performance. The Pocock boundary may be replaced by the ad hoc boundary for the secondary endpoint with a very little loss of secondary power if the practical concern is at issue. A clinical trial example is given to illustrate the methods.     

Vegetation as a component of a non-nested hierarchy: a conceptual model

Abstract. A general conceptual model of vegetation based on hierarchy theory is presented. The model emphasizes that prediction of vegetation requires consideration of both mechanisms of vegetation change and the constraints within which it occurs. The mechanisms of vegetation change are the responses to and effects upon their surroundings of individual plants. The most general constraints upon vegetation are aspects of the environment not affected by vegetation over successional time, and the pool of species within dispersal range. Examples of such environmental factors include macroclimate and soil parent material. In some cases, vegetation may alter important labile environmental factors such as soil nutrient and water availability. Some vegetation compositions appear to be resistant to changes in the general constraints. Due to both sources, there are multiple possible vegetation compositions given the same general constraints. Disturbance is defined as an abrupt change in the constraints on the vegetation resulting in a change in the vegetation's state or dynamics. Both the recognition of disturbance and the distinction between independent and labile environmental factors depend on the spatial and temporal scale of observation. For example, a particular wildfire at a given stand may be a disturbance, whereas at a larger scale of observation the same event may contribute to the wildfire regime, part of the constraints at that scale. Similarly, levels of soil organic matter may constrain vegetation over short time scales, due to influencing availability of water and nutrients. Over long time scales, the vegetation itself is a primary determinant of soil organic matter content. This model contains elements of both the initial, holistic theory of vegetation and recent, reductionistic approaches. It reiterates the need to considerboth mechanisms and constraints, stressed by contemporary and earlier workers. Hierarchy theory provides new insights concerning sufficient conditions for prediction, possible limits on predictability, and appropriate research strategy.     

Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.     

Heating of a Local Region of a Branching Streamer as a Starting Point of a Space Leader and a Negative-Leader Step

Plasma Physics Reports - Localized plasma formations called space leaders are observed in streamer coronas of negative leaders of long laboratory sparks. The main leader completes a step when the...     

From a homeostatic to a homeodynamic self

Life as an autonomous homeostatic system is discussed. A mechanism that drives a homeostatic state to an autonomous self-moving state is examined with two computational cell models. The mechanism is met with Ashby's ultrastability, where random parameter searching is activated when a system breaks a viability constraint. Such a random search process is replaced by the membrane shape in the first model and by chaotic population dynamics in the second model. Emergence of sensors, motors and the recursive coupling between them is shown to be a natural outcome of an autonomous homeostatic system.