DNA frayed wires: Differential polymerization of d(AnGm) oligonucleotides

Oligodeoxyribonucleotides with terminal runs of contiguous guanines, d(A_nG_m), spontaneously associate into high molecular weight complexes that resolve on polyacrylamide gels as a regular ladder pattern of bands with low mobility. The aggregates, which we call frayed wires, arise from the interaction between the guanine residues of the oligonucleotides; the adenine tracts are single stranded and can take part in Watson–Crick interactions. Oligonucleotides, with different arm‐to‐stem ratios and total length, readily associate in the presence of Mg²⁺ to form aggregates consisting of an integer number of strands. The type of the observed aggregates is determined by the length of the guanine run. Oligonucleotides with six guanines form four‐ and eight‐stranded complexes; there is no further polymerization. An increase in the number of guanine residues to 10 and 15 leads to polymerization resulting in a ladder pattern of up to 9 bands and an intense signal at the top of the gel. The relative population of any given species in a frayed wire sample is governed by the guanine stem length and is not affected to any substantial extent by arms up to 40 bases long. The type and concentration of the cation in the solution affect the degree of aggregation, with Na⁺ and K⁺ promoting the formation of complexes comprised of 2–4 strands and Mg²⁺ being the most effective in facilitating polymerization. The electrophoretic behavior of frayed wires was analyzed in the framework of the Ogston theory. The free mobility of frayed wires in the solution is close to the values reported for single‐stranded DNA, indicating the equivalence of the charge density of the two conformations. The retardation coefficients for frayed wires arising from a single kind of parent strand increase with the introduction of each additional strand. There is no correlation between the retardation coefficient and the type of parent strand; rather, the magnitude of the retardation coefficient is determined by the total molecular weight of the complex. The values of the retardation coefficients are consistently higher than those for double‐stranded DNA and they display much stronger dependence on the total molecular weight. Presumably, the distinct structural and dynamic characteristics of the two conformations account for their different electrophoretic behavior. © 1999 John Wiley & Sons, Inc. Biopoly 49: 287–295, 1999     

Electrohydrodynamic instabilities and segregation of polysaccharides in capillary polymer solution electrophoresis

During capillary electrophoresis of negatively charged polysaccharides in polymer solutions as sieving media, concentration fluctuations develop due to electrohydrodynamic instabilities caused by polarization of the polyelectrolytic chains. This leads to deviations from electroneutrality far beyond the Debye layer and segregation of the initially homogeneous sample solution into aggregated sample‐rich domains as verified by epifluorescence videomicroscopy imaging. As a result, anomalous and irregular peak profiles are obtained impeding the characterization of such complex sample mixtures. This effect appears at an electric field strength threshold value that depends on the molecular weight of the solute polymer molecules, pH, type and concentration of the polymer solution sieving media, and buffering conditions. The magnitude increases with increasing field strength and amount of sample injected. The aggregation onset, as evaluated by the value of the threshold potential, is affected by the charge density of the sample polymer molecules and Debye screening effects and investigated through variation of pH and ionic strength, respectively. Exchange of a simple base buffer component for small and multiply charged organic bases markedly increases the electric field strength necessary to trigger the electrohydrodynamic instabilities. Ultimately, the threshold value could be increased more than seven times by addition of an oppositely charged aminodextran polymer, thereby decreasing the analysis time. © 1999 John Wiley & Sons, Inc. Biopoly 49: 515–524, 1999     

家蚕蜕皮液蛋白质双向电泳及质谱分析

蜕皮液是存在于新旧表皮之间的一层液体,在昆虫蜕皮和变态发育的过程中发挥了重要的作用。为进一步探究家蚕蜕皮液的功能,利用双向电泳技术对家蚕预蛹期及羽化前期的蜕皮液的蛋白质进行了分析,结果表明,预蛹期及羽化前期的蜕皮液中分别可以检测出超过200个蛋白点,它们主要分布在等电点4-9、分子量10-180 kDa之间。利用MALDI TOF/TOF对羽化前期蜕皮液的42个蛋白点进行了鉴定分析,结果表明34个蛋白点成功得到了鉴定,它们主要包括载脂蛋白类、蛋白酶与蛋白酶抑制剂、免疫相关蛋白、几丁质结合蛋白等,部分蛋白在预蛹期的蜕皮液和羽化前的蜕皮液之间存在明显的差异表达。为了进一步验证蛋白质组分析的结果,对其中1个差异表达明显的蛋白质Apolipoprotein D进行了进一步的分析,Q-PCR的结果表明,该蛋白主要在化蛹第1–4天存在高表达,其在羽化前蜕皮液中的高度累积暗示了它可能参与了家蚕羽化变态的过程。以上研究结果进一步丰富了人们对蜕皮液蛋白质的认识,为深入研究蜕皮液蛋白质的功能提供了一些参考。     

Assessment of sample preparation methods for metaproteomics of extracellular proteins

Enzyme discovery in individual strains of microorganisms is compromised by the limitations of pure culturing. In principle, metaproteomics allows for fractionation and study of different parts of the protein complement but has hitherto mainly been used to identify intracellular proteins. However, the extracellular environment is also expected to comprise a wealth of information regarding important proteins. An absolute requirement for metaproteomic studies of protein expression, and irrespective of downstream methods for analysis, is that sample preparation methods provide clean, concentrated and representative samples of the protein complement. A battery of methods for concentration, extraction, precipitation and resolubilization of proteins in the extracellular environment of a constructed microbial community was assessed by means of 2D gel electrophoresis and image analysis to elucidate whether it is possible to make the extracellular protein complement available for metaproteomic analysis. Most methods failed to provide pure samples and therefore negatively influenced protein gel migration and gel background clarity. However, one direct precipitation method (TCA-DOC/acetone) and one extraction/precipitation method (phenol/methanol) provided complementary high quality 2D gels that allowed for high spot detection ability and thereby also spot detection of less abundant extracellular proteins.     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

The Arabidopsis trichome is an active mechanosensory switch

Trichomes (‘hair cells’) on Arabidopsis thaliana stem and leaf surfaces provide a range of benefits arising from their shape and disposition. These include tempting herbivores to sample constitutive toxins before they reach the bulk of the tissue. We asked whether, in addition, small mechanical disturbances such as an insect can make elicit signals that might help the plant respond to herbivory. We imaged, pressed and brushed trichomes in several ways, most notably with confocal microscopy of trichomes transgenically provided with apoplastic pH reporter apo‐pHusion and cytosolic Ca²⁺ reporter cameleon. In parallel, we modelled trichome wall mechanics with finite element analysis. The stimulated trichome focuses force on a pliant zone and the adjoining podium of the stalk. A buckling instability can further focus force on a skirt of cells surrounding the podium, eliciting oscillations of cytosolic Ca²⁺ and shifts in apoplastic pH. These observations represent active physiological response. Modelling establishes that the effectiveness of force focusing and buckling is due to the peculiar tapering wall structure of the trichome. Hypothetically, these active mechanosensing functions enhance toxin synthesis above constitutive levels, probably via a priming process, thus minimizing the costly accumulation of toxins in the absence of herbivore attack but assuring rapid build‐up when needed.     

甘蓝型油菜种子中油体的超微结构及蛋白质组分析

以甘蓝型油菜（Brassica napus L.）含油量较高的品种‘ZS11’、含油量中等的品种‘Westar’和‘Topas’以及含油量较低的品种‘ZS10’为实验材料,通过超微结构观察和统计,比较分析不同品种种子中油体形态、大小和数量的差异。研究结果显示,品种‘ZS11’种子子叶细胞油体排列致密,形态较小,大部分油体的直径低于1 μm;而在含油量中等或较低的品种中,种子子叶细胞油体排列均显疏松,其中‘Westar’和‘Topas’的油体较大,而‘ZS10’的油体大小不一。本研究还通过双向电泳分析进一步检测了‘Westar’和‘ZS11’种子中总蛋白和油体蛋白的差异表达情况。结果显示,‘Westar’和‘ZS11’种子总蛋白双向电泳图谱中,表达量具有2倍以上差异的蛋白质点共有57个;其中在‘Westar’中特异表达的种子总蛋白质点有24个,在‘ZS11’中有23个。在上述2个品种油体蛋白双向电泳图谱中,表达量具有2倍以上差异的蛋白质点共有52个,在品种‘Westar’中特异表达的有2个,‘ZS11’中有13个。表明不同含油量的油菜品种种子在油体的结构和蛋白组份上均存在差异。     

硫素对不同大豆品种成熟期AM真菌群落多样性的影响

研究硫素对不同大豆品种成熟期丛枝菌根（arbuscular mycorrhizal,AM）真菌群落多样性的影响,探索有利于提高3个特定大豆品种根围土壤和根系AM真菌多样性的最佳施硫量,为提高大豆产量和改善大豆品质提供理论依据。试验采用盆栽,选用黑农44（HN44）、黑农48（HN48）、黑农37（HN37）3个大豆品种作为试验材料,设4个硫素处理S1（对照）,S2（0.02g/kg）,S3（0.04g/kg）和S4（0.06g/kg）。采用PCR-DGGE技术分析3个大豆品种根围土壤和根系中AM真菌群落多样性。结果表明：在S2处理下HN37和HN44根围土壤和根系AM真菌多样性最高,而在S3处理下HN48根围土壤和根系AM真菌多样性最高;DGGE图谱中各样品优势种群变化显著,球囊霉属Glomus和柄囊霉属Funneliformis真菌为3个大豆品种根围土壤和根系中AM真菌的优势菌群。由此可见,硫素对不同大豆品种根围土壤和根系AM真菌群落多样性有显著影响,适量施硫能够提高大豆根围土壤和根系中AM真菌的多样性,不施或过高施硫反而抑制AM真菌的多样性。     

盐胁迫下棉花基因组基于毛细管电泳的MSAP分析

以棉花杂交种中棉所29为材料,用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism,MSAP) 分析法结合毛细管电泳检测技术进行甲基化鉴定,以初步探讨棉花耐盐的分子机理．应用24个引物组合,中棉所29在0.4%盐水胁迫及清水对照下,平均每引物组合检测甲基化位点数分别为69.2和56.7,差异达显著水平．盐胁迫下的DNA甲基化水平与清水对照下相比,52.6%位点表现出甲基化水平提高,即发生了超甲基化;19.7%位点甲基化水平降低,即表现为次甲基化;二者差异达极显著水平．研究结果表明,中棉所29盐胁迫后发生了广泛的DNA甲基化变化,包括超甲基化和次甲基化,以及其它甲基化类型的转变|发生超甲基化位点极显著地多于发生次甲基化位点．盐胁迫下的中棉所29与对照相比,DNA总体甲基化水平显著提高,暗示中棉所29有提高基因组甲基化水平以应对盐胁迫的潜在机制,棉花基因组整体甲基化水平的提高可能与棉花对盐胁迫的耐受性起重要作用．本研究中,甲基化序列的初步克隆及比对分析表明,盐胁迫前后多个ATP合成相关基因甲基化程度维持在同一水平,其表达不受甲基化影响,这也可能是中棉所29耐盐性较强,在一定时间盐处理后能维持正常生长的原因之一．     

Isolation,purification and physicochemical characterization of water‐soluble Bacillus thuringiensis melanin

Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water‐insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water‐soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water‐soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water‐soluble melanin from this organism has an isoelectric point (pI = 3.0–3.2) and was purified optimally by adsorbtion using the IA‐1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra‐red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS‐PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g = 2.006. The concentration of paramagnetic centers in melanin is 0.21 × 10¹⁸ spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.