一种基于特征筛选的原核生物启动子判别分析方法

启动子识别是研究基因转录调控的重要环节,但目前方法的识别正确率偏低。在深入分析原核启动子特征的基础上,提出了一种基于特征筛选的原核启动子判别分析方法,首先在启动子序列的组成特征、信号特征和结构特征中选取备选特征,为每个特征建立适当的描述模型,并对主要的保守模式采用复合模式模型;再通过模型计算对备选特征进行逐步筛选,优化特征集,将序列表示为组合特征向量;最终利用二次判别分析实现识别。对大肠杆菌和枯草杆菌实际启动子数据进行的刀切法测试验证了方法的有效性和通用性。对于大肠杆菌非编码区(70启动子,识别的平均正确率达到了85.8%,优于其它几种典型识别方法;对于大肠杆菌编码区内部)70启动子和其它几种原核启动子,平均正确率也都超过了80%。方法框架还具有良好的可扩展性,能够方便地容纳新特征,使识别性能不断提高。     

原核启动子文库构建的研究进展

启动子是在转录水平上调节基因表达的关键元件，启动子文库广泛应用在代谢工程和合成生物学专业中，主要用于调节基因表达和优化代谢物生物合成。运用启动子对多基因通路中的基因表达进行合理和精确控制，可显著影响代谢通量分布，优化特定目标代谢物的合成。因此，国内外已有大量关于鉴定天然启动子，构建和设计组成型、诱导型、杂交启动子或人工合成启动子文库的研究。文章简要介绍原核启动子的结构和功能，对基于生物信息学的启动子预测和识别及人工构建启动子文库策略的最新进展进行了归纳总结，以期为原核微生物的代谢工程和合成生物学应用提供帮助。     

原核表达系统T7RNA聚合酶/启动子在真核细胞中表达目的基因的实验研究

将目前高表达水平强大的原核表达系统之一T7RNA聚合酶 启动子表达系统通过一系列改进引入真核细胞。通过转染真核细胞实验表明 ,采用真核启动子CMV调控T7RNA聚合酶的表达和在T7启动子下游插入EMCVIRES序列两种解决方案能使该原核表达系统在真核细胞高效表达目的基因 ,且能适应不同的真核细胞环境 ,是一良好的细胞类型非依赖的表达体系。     

以β-半乳糖苷酶为报告基因的原核启动子检测体系构建

目的:以β-半乳糖苷酶为报告基因构建原核启动子检测体系.方法:以由质粒 pDL 扩增获得的 bgaB 基因为报告基因,将其克隆到大肠杆菌-枯草杆菌穿梭载体 pBE2,构建成一个具有启动子活性检测功能的重组质粒pBEB.将组成型启动子P43和诱导型启动子Pcpac克隆入 pBEB,得到重组表达载体 pBEBP43 和 pBEBPapac,转化至大肠杆菌和枯草芽孢杆菌.结果:两种不同类型的启动子均能在大肠杆菌 BL21 和枯草芽孢杆菌 1A751 中启动 bgaB 基因的表达.结论:成功构建具有较为广泛适用性的原核启动子检测体系.     

原核表达系统T7RNA聚合酶/启动子在真核细胞中表达目的基因的实验研究

将目前高表达水平强大的原核表达系统之一T7 RNA聚合酶/启动子表达系统通过一系列改进引入真核细胞.通过转染真核细胞实验表明,采用真核启动子CMV调控T7 RNA聚合酶的表达和在T7启动子下游插入EMCV IRES序列两种解决方案能使该原核表达系统在真核细胞高效表达目的基因,且能适应不同的真核细胞环境,是一良好的细胞类型非依赖的表达体系.     

具有原核基因启动子活性的蓖麻蚕染色体DNA片段

利用大肠杆菌启动子克隆载体pHE5,研究了蓖麻蚕染色体的Hind Ⅲ酶解片段在大肠杆菌中作为四环素抗性基因启动子的功能作用。蓖麻蚕染色体的Hind Ⅲ片段与pHE5重组,在大约10~6个重组子中获得953株启动子重组体。测定了这些含有重组质粒的菌株抗四环素能力,其中有4株在平板上抗四环素水平超过225微克/毫升。对其中pARP201的插入片段进行了限制性图谱分析和启动子活性区域的缺失定位,证明了pARP-DB(由pARP201衍生而来)中的约0.5kb的外源插入片段具有完整的原核启动子功能。我们分析了这段DNA的部分核苷酸顺序,发现它与原核基因启动子极其相似。     

cⅠ857基因的体外定位同义突变

 ｃⅠ８５７基因的体外定位同义突变陈南春,高辉,陈苏民,杨萍,刘新平（西安第四军医大学分子生物学研究所,西安７１００３２）外源基因要在大肠杆菌中获得高表达,需要合适的ＳＤ序列和可调控的强启动子［１］。ＰＬ启动子在原核启动子中属强启动子,它受ｃⅠ基因产物...     

枯草芽孢杆菌表达系统及其启动子研究进展

枯草芽孢杆菌作为一种革兰氏阳性细菌,由于其具有非致病性、分泌蛋白能力强的特性和良好的发酵基础及生产技术,是目前原核表达系统中表达和分泌外源蛋白的理想宿主,成为原核表达系统中的一种重要的模式菌株。而实现外源蛋白的高效表达的关键因素之一是使用强并可控制的启动子。目前,枯草芽孢杆菌中常用的启动子为组成型、诱导物诱导型、时期特异性及自诱导型。详细介绍枯草芽孢杆菌表达系统以及其常用启动子的优缺点,并对克隆新的启动子的方法做了总结,旨为完善枯草表达系统和工业生产外源蛋白奠定基础。     

一种基于lacZ基因和pUC复制子的原核启动子报告系统的构建及评价

为构建一种具有广泛适用性的原核启动子报告系统,以质粒pFLX107为骨架,通过多克隆位点替换和序列改造,构建出基于lacZ基因和pUC复制子的pFGH系列报告载体,然后以lacZ基因缺失株MC4100为宿主菌筛选背景活性最低的质粒作为最终的报告系统,并利用诱导型启动子araBAD和组成型启动子rpsM分别对其进行测试。结果显示,在所构建的pFGH系列质粒中,pFGH06的背景活性显著低于同系列其他质粒,在28 ℃培养条件下甚至显著低于低拷贝参考质粒pRCL的活性 (P<0.01)。进一步的评估测试显示,质粒pFGH06可用于诱导型启动子或组成型启动子的克隆及活性测定,且在模拟应用于启动子筛选时,通过蓝白斑筛选即可实现对目标启动子的完全识别。与已报道的原核启动子报告系统相比,pFGH06具有体积小、克隆位点多、背景活性可调、对启动子筛选识别效率高等优点,具有广泛的应用前景。     

含PR启动子的原核高效表达载体的构建及应用

组建了一个仅含PR启动子的原核高效表达载体pRC,它同时含有cⅠ调控基因、多酶切位点和两个强的转录终止序列.现已成功地用于表达重组人肿瘤坏死因子-α(hTNF-α)、重组人白细胞介素-3(hIL-3)和抗溶菌酶(HEL)抗体Fd基因,表达量均占菌体总蛋白的36%以上.同时还研究了不同的宿主菌和原核增强子序列等因素对PR启动子载体表达的影响.此外,还比较了分别以PR、PL或PRPL作为启动子时表达hTNF-α的情况,结果表明,单用PR或PL启动子可获得与使用PRPL串联启动子一样的高效表达.     

An Ecosystem Health Index for a large and variable river basin: Methodology,challenges and continuous improvement in Queensland’s Fitzroy Basin

Report cards are an increasingly popular method for summarising and communicating relative environmental performance and ecosystem health, including in aquatic environments. They are usually underpinned by an Ecosystem Health Index (EHI) that combines various individual indicators to produce an overall ecosystem health “score”. As a result of public water quality concerns, an integrated means of monitoring and reporting on aquatic ecosystem health was needed for the Fitzroy Basin in central Queensland, Australia. The Fitzroy Partnership for River Health was formed to address this need, and developed an EHI and report card for the Basin using existing monitoring data collected from various third parties including regulated companies operations and government. At 142,000 square kilometres, the Fitzroy Basin is the largest catchment draining to the World Heritage Listed Great Barrier Reef. The Fitzroy Basin provides an example of how to deliver an effective aquatic ecosystem health reporting system in a large and complex river basin. We describe the methodology used to develop an adaptive EHI for the Fitzroy Basin that addresses variability, complexity and scale issues associated with reporting across large areas. As well, we report how to manage the design and reporting stages given limitations in data collection and scientific understanding.     

New strigolactone mimics: Structure–activity relationship and mode of action as germinating stimulants for parasitic weeds

Strigolactones (SLs) are new plant hormones with varies important bio-functions. This Letter deals with germination of seeds of parasitic weeds. Natural SLs have a too complex structure for synthesis. Therefore, there is an active search for SL analogues and mimics with a simpler structure with retention of activity. SL analogues all contain the D-ring connected with an enone moiety through an enol ether unit. A new mechanism for the hydrolysis SL analogues involving bidentate bound water and an α,β-hydrolase with a Ser-His-Asp catalytic triad has been proposed. Newly discovered SL mimics only have the D-ring with an appropriate leaving group at C-5. A mode of action for SL mimics was proposed for which now supporting evidence is provided. As predicted an extra methyl group at C-4 of the D-ring blocks the germination of seeds of parasitic weeds.     

Social Relations and Commodity Chains: The Live Reef Fish for Food Trade

This paper considers the relationship between anthropology and the interdisciplinary field of commodity studies through an examination of an important commodity chain in the Asia-Pacific, the live reef fish for food trade. Using an ethnographic perspective, I focus on how particular social relationships within this commodity chain have significant implications for two key concerns of commodity studies—the distribution of benefits through commodity chains and how commodity chains are regulated. The social relationships between fishers and traders provide a powerful avenue for relative economic prosperity for fishers, yet the forms of social relationships that operate between fishers, traders and regulators mean that regulation of the trade is not being implemented. This analysis provides an example of how an ethnographic lens can provide a useful perspective that can contribute to an interdisciplinary dialogue on commodity chains.     

Physical Attractiveness of an Animal Species as a Decision Factor for its Preservation

ABSTRACT

This experimental study examines how framing an animal species as endangered affects its perceived attractiveness and how an animal's perceived attractiveness affects support for its conservation. Undergraduate students were shown a flyer from a fictitious environmental organization pleading for the protection of either a bat or an ape. The flyer contained either no picture, a picture of an attractive member of its species, or a picture of an unattractive member of its species. For both species, an animal's attractiveness substantially increased support for its protection. There was also more support for saving the species that was larger in size and more resembled humans. In a second experiment, participants rated an unattractive animal as more attractive if it was framed as endangered. There was no such effect for an animal originally perceived as attractive. The implications of the results for environmental policy and conservation support are discussed.     

Application Fields of the Empirical Regression - A Case Study

Empirical regression is defined as conditional expected value based on an estimation of a twodimensional density. It is a modelfree mathematical means for a first evaluation of measured data of an unknown stochastical relation between two quantities. The numerical procedures may be applied for calculation of the mean course of an unknown relation hidden in the measured data. Disregarding the statistical background an other aspect of application is the analyzing of time series, especially smoothing of time series and modelfree recording of the trend component in non-stationary time series. The calculated regression curve provides an objective basis for comparing of different measured courses as well as for a further evaluation, e. g. in respect of a suitable choice of an analytical expression. The possibility of interpolation and the smoothing properties of empirical regression give essential advantages for internal regression as one step in the process of model construction.     

Design and management of an orthopaedic bone bank in the Netherlands

The design and management of an orthopaedic bone bank is a complex process in which medical organisation and legislation intertwine. Neither in the Netherlands, nor in any other European country, there are official guidelines for the organisation and management of an orthopaedic bone bank. In the Netherlands, the recently modified ‘law of security and quality for using human materials’ (WVKL) dictates requirements for technical and organisational aspects for the use of human tissue and cells. The bone bank procedures include a thorough questionnaire for donor selection, extensive serological, bacteriological and histopathological examination, as well as standard procedures for registration, processing, preservation, storage and distribution of bone allografts. This article describes the organisation of an accredited bone bank and can be used as a proposition for an official guideline or can be useful as an example for other orthopaedic bone banks in Europe.     

Toward quantification of protein backbone-backbone hydrogen bonding energies: An energetic analysis of an amide-to-ester mutation in an alpha-helix within a protein

Amide-to-ester backbone mutagenesis enables a specific backbone-backbone hydrogen bond (H-bond) in a protein to be eliminated in order to quantify its energetic contribution to protein folding. To extract a H-bonding free energy from an amide-to-ester perturbation free energy (DeltaG (folding,wt) - DeltaG (folding,mut)), it is necessary to correct for the putative introduction of a lone pair-lone pair electrostatic repulsion, as well as for the transfer free energy differences that may arise between the all amide sequence and the predominantly amide sequence harboring an ester bond. Mutation of the 9-10 amide bond within the V9F variant of the predominantly helical villin headpiece subdomain (HP35) to an ester or an E-olefin backbone bond results in a less stable but defined wild-type fold, an attribute required for this study. Comparing the folding free energies of the ester and E-olefin mutants, with correction for the desolvation free energy differences (ester and E-olefin) and the loss of an n-to-pi* interaction (E-olefin), yields an experimentally based estimate of +0.4 kcal/mol for the O-O repulsion energy in an alpha-helical context, analogous to our previous experimentally based estimate of the O-O repulsion free energy in the context of a beta-sheet. The small O-O repulsion energy indicates that amide-to-ester perturbation free energies can largely be attributed to the deletion of the backbone H-bonds after correction for desolvation differences. Quantitative evaluation of H-bonding in an alpha-helix should now be possible, an important step toward deciphering the balance of forces that enable spontaneous protein folding.     

Aggregation kinetics of extended porphyrin and cyanine dye assemblies

The kinetics of J-aggregate formation has been studied for two chromophores, tetrakis-4-sulfonatophenylporphine in an acid medium and pseudoisocyanine on a polyvinylsulfonate template. The assembly processes differ both in their sensitivity to initiation protocols and in the reaction profiles they produce. The porphyrin's assembly kinetics, for example, displays an induction period unlike that of the cyanine dye. Two kinetic models are presented. For the porphyrin, an autocatalytic pathway in which the formation of an aggregation nucleus is rate-determining appears to be applicable; for the pseudoisocyanine dye, an equation derived for diffusion-limited aggregation of a fractal object satisfactorily fits the data. These models are shown to be useful for the analysis of kinetic data obtained for several biologically important aggregation processes.     

Advanced Liposomal Vectors as Cancer Vaccines in Melanoma Immunotherapy

Malignant tumors represent a major source of disability and account for more than one of five deaths in Western countries. Among the different cancers, melanoma harbors two distinctive features. First, its has long been recognized as an immunogenic tumor, and second, an unprecedented rise in incidence is currently observed, in face of few therapeutic options. Thus, melanoma represent an ideal target for a cancer immunotherapy program. To date, a number of immunodominant epitopes from tumor associated antigens (TAA) are used as cancer vaccines in clinical trials, in spite of an acknowledged rapid degradation in vivo and low immunogenicity. However, most of the immunotherapy trials reported so far do not achieve consistent clinical results. Hence, there is an urgent need for the development of a carrier system and strong adjuvants suitable for a TAA-based cancer immunotherapy. Liposomes and their further development as virosomes with added adjuvancy may address both these issues. We report here our experience in the tailoring of dedicated advanced liposomal vectors that were developed in the context of an upcoming immunotherapy clinical trial for melanoma.     

Hydrodynamic cavitation as a strategy to enhance the efficiency of lignocellulosic biomass pretreatment

Hydrodynamic cavitation (HC) is a process technology with potential for application in different areas including environmental, food processing, and biofuels production. Although HC is an undesirable phenomenon for hydraulic equipment, the net energy released during this process is enough to accelerate certain chemical reactions. The application of cavitation energy to enhance the efficiency of lignocellulosic biomass pretreatment is an interesting strategy proposed for integration in biorefineries for the production of bio-based products. Moreover, the use of an HC-assisted process was demonstrated as an attractive alternative when compared to other conventional pretreatment technologies. This is not only due to high pretreatment efficiency resulting in high enzymatic digestibility of carbohydrate fraction, but also, by its high energy efficiency, simple configuration, and construction of systems, besides the possibility of using on the large scale. This paper gives an overview regarding HC technology and its potential for application on the pretreatment of lignocellulosic biomass. The parameters affecting this process and the perspectives for future developments in this area are also presented and discussed.