短DNA片段缩合结构的比较研究

本文利用透射式电镜对四种短DNA片段(500、1100、1500、2700 bP)的缩合结构进行了比较研究得出很有意义的结果。定量研究证实短至500 bP的DNA分子仍可形成复曲面,且分子量相差5倍多的DNA片段缩合形成的复曲面尺度大小一致。复曲面外径为400A左右。从而进一步证实作者与Arscott及Bloomfield关于复曲面尺度独立于DNA分子量,及短DNA片段的缩合是多分子缩合的结论。此外,观测到缩合中间结构的尺度依DNA分子量大小不同而变化,同时分子量愈小的DNA片段产生另一种缩合结构—棒体的几率愈大。     

短DNA片段缩合结构的比较研究

本文利用透射式电镜对四种短DNA片段(500、1100、1500、2700 bP)的缩合结构进行了比较研究得出很有意义的结果。定量研究证实短至500 bP的DNA分子仍可形成复曲面,且分子量相差5倍多的DNA片段缩合形成的复曲面尺度大小一致。复曲面外径为400A左右。从而进一步证实作者与Arscott及Bloomfield关于复曲面尺度独立于DNA分子量,及短DNA片段的缩合是多分子缩合的结论。此外,观测到缩合中间结构的尺度依DNA分子量大小不同而变化,同时分子量愈小的DNA片段产生另一种缩合结构—棒体的几率愈大。     

提高易自聚合蛋白质与核酸相互作用效率的一种简单方法

对蛋白质、DNA和RNA的相互作用(结合)的研究是分子生物学的基本课题.多数DNA和RNA的结合蛋白都具有自聚合倾向,在体外实验中会造成难以和DNA或RNA形成结合物而影响实验的结果.用荧光素异硫氰酸酯(FITC)标记蛋白质能显著抑制这种自聚合倾向,而大幅度提高其与核酸分子的结合效率.这一简单方法已用于在细胞角蛋白18与转录因子C/EBPβ3′UTR RNA结合研究中.     

DNA计算的规律及其在解题中的应用

归纳了DNA计算题的解题规律．如DNA分子中碱基数目的计算规律、DNA复制所消耗的脱氧核苷酸数的计算规律、基因指导蛋白质合成的计算规律、脱氧核苷酸脱水缩合的规律、碱基排列顺序数的计算规律和DNA分子中氢键数的计算规律等及其在解题中的应用。     

人神经tau蛋白与DNA相互作用

通过凝胶阻滞实验表明,人类神经tau能够与不同来源的DNA（λDNA, 质粒DNA以及PCR产物）相结合,形成tau-DNA复合物.每分子神经tau大约与长度为6～10 bp的核苷酸片段结合.原子力显微镜直接证实了tau与线性质粒DNA相结合形成串珠样的结构.采用单克隆抗体Tau-1,进行免疫组织化学的实验显示,神经tau不但弥散地分布在SY5Y细胞系的细胞质内,同时也存在于细胞核中,并形成染色致密的斑点.以上结果提示：神经tau在细胞核中可能参与了某种重要的生物学功能.     

科学家成功合成长链RNA分子

数亿年前DNA和RNA分子如何从简单的单体聚合而成,一直是关于生物分子起源的一个难题,近日,意大利的研究人员重建了这一过程,仅仅在温水的反应条件下就使得简单的核苷酸聚合成了氏链的RNA分子。     

抗稻瘟病水稻资源抗性基因Pita、Pib、Pi9以及Pikm的分布研究

利用抗稻瘟病水稻资源品种杂交,聚合多个抗性基因是培育持久抗稻瘟病水稻新品种的主要育种途径.利用分子标记技术对水稻抗性资源进行基因型鉴定是分子辅助聚合育种的基础.通过以亚华种业科学院稻瘟病病圃抗病水稻资源为材料,利用特异性分子标记对Pi9、Pita、Pib以及Pikm基因在水稻抗稻瘟病资源的分布进行了鉴定,初步建立了抗性基因数据库.同时对抗性基因及与抗性反应的相关性进行了探讨,结果表明以Pi9为主效基因,同时聚合Pita和Pib抗性基因能提高持久抗稻瘟病能力.     

DNA自由能与DNA空间几何构型关系研究

对DNA空间几何结构的研究,通常是把DNA螺旋轴当作空间扭转曲线,使用弹性杆模型进行研究.本文以描述DNA构型转换的Landau(朗道)模型和微分几何方法为基础,研究了DNA自由能与DNA空间几何构型关系.首先我们介绍了描述DNA的Landau模型.然后将DNA的螺旋轴当做空间扭转曲线,建立Frenet标架,得到了空间处曲线的曲率.而实际上DNA螺旋轴是处在DNA的扭转曲面上的,它是存在于曲面上的曲线.考虑到曲面的特性,我们重新定义了曲线上三维正交标架,并把螺旋线当做当做曲面上的曲线进行研究:首先建立空间曲面上扭转曲线的Frenet标架,参考空间曲线的Frenet公式得到空间曲面上所满足的方程组.其次,由此方程组经过相关欧拉角的运算,得到了DNA双螺旋扭转曲面上螺旋轴的曲率和挽率.最后,联系朗道模型得到以DNA双螺旋扭转曲面上的螺旋轴的曲率和挠率来表示的DNA自由能的简洁表达.通过得到的DNA自由能与DNA其空间几何结构的关系式我们可以直观的了解到DNA的自由能是由DNA双螺旋扭转曲面上螺旋轴的曲率和挠率所决定的:当DNA链的扭转曲面上的曲率和挠率确定时,DNA的自由能就能被完全的确定下来;同样,如果通过能量的改变,也可以确定DNA空间几何结构不同构象.为以后的DNA自由能以及结构的研究做出了参考性的意见.     

多价阳离子诱导的DNA缩合研究进展

DNA分子在多价阳离子作用下,可以缩合成紧密有序的纳米级缩合体。常用的多价阳离子包括阳离子聚合物,蛋白质,高价无机离子等。缩合主要是由于67～90%的DNA磷酸基负电荷被中和而引起的,缩合的过程伴随着微粒的聚集过程;常见的缩合体是圆环体状,其尺寸一般在50～300nm之间;缩合主要受缩合剂正电荷的影响,缩合剂的结构也对缩合过程有一定的影响;DNA分子的大小和碱基成分也是影响缩合的因素,同时也决定缩合体的体积大小。     

甲基绿(Methyl green)及焦宁(Pyronin)与DNA的结合作用

(1)小牛胸腺DNA溶液在100℃保温不同时间以后,测定了粘度的变化,同时观察并比较甲基绿与焦宁对于受热变性前后的DNA的结合能力的差异。(2)根据高分子物质与低分子物质结合作用的一般规律,测定了甲基绿对于天然态DNA及数种热变性DNA的结合数据。(3)论证了甲基绿对DNA的结合能力主要是与DNA分子的双螺旋空间构型的完整性有关。(4)对甲基绿与DNA结合的机制作了初步的讨论。(5)从甲基绿与焦宁对DNA的双重结合作用所得结果表明:两种染料能以不同程度同时与DNA结合,但在此两者之间存在着一定程度的竞争作用,以甲基绿占明显的优势。从所得结果以及两种染料的分子结构的差异可猜测,此两种染料对于DNA分子的空间构型的要求程度不同。     

Condensation of DNA by trivalent cations. 1. Effects of DNA length and topology on the size and shape of condensed particles

In vitro condensation of DNA by multivalent cations can provide useful insights into the physical factors governing folding and packaging of DNA in vivo. We have made a detailed study of hexammine cobalt (III) induced condensation of 2700 and 1350 base pair (bp) fragments of plasmid pUC12 DNA by electron microscopy and laser light scattering. The condensed DNA takes the form of toroids and rods. Both are present in all condensates, but the proportion of toroids is higher with the larger fragments. The intact, closed circular plasmid produces smaller particles than the linear fragments. The size of a particle is independent of DNA fragment length. Two hours after adding the condensing agent, a typical toroid is about 800 A in diameter; the outer radius (R1) is approximately 400 A, and the inner radius (R2) is approximately 140 A for both sets of fragments. These dimensions are relatively stable, but there is sufficient change in both R1 and R2 to produce approximately 50% increase in volume from 2 to 24 h. A typical rod at 2 h is about 1800 A long and 300 A wide. The distribution of rod lengths is similar to that of mean toroid circumferences pi (R1 + R2), and the distribution of rod widths is similar to that of toroidal widths (R1-R2). The 2700-bp fragments show a significantly higher ratio of toroids to rods than the 1350-bp fragments. Both types of particle are multimolecular. The average number of molecules/particle, calculated from the above dimensions, assuming hexagonally packed B-form DNA with a center-to-center spacing of 27 A, is 13 +/- 4 for condensates of 2700-bp fragments and 26 +/- 11 for those of 1350-bp fragments. Monomolecular condensates of much larger DNAs have similar dimensions, suggesting that size is governed primarily by the balance of attractive and repulsive intermolecular forces rather than by the entropic factors associated with incorporation of a number of small particles into a larger one. The similar dimensions and volumes of toroids and rods indicate that the free energy cost of continual bending in toroids, minus that gained by extra net attraction in a cyclic particle, is comparable to that of abrupt bending or kinking in rods. Although the condensed particles are multimeric, their distinct toroidal or rodlike shapes distinguish them from the random aggregates that would be generally expected from the multimolecular association of large, flexible polymers.     

Influence of DNA length on spermine-induced condensation. Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

Influence of DNA length on spermine-induced condensation: Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

Time study of DNA condensate morphology: implications regarding the nucleation, growth, and equilibrium populations of toroids and rods

It is well known that multivalent cations cause free DNA in solution to condense into nanometer-scale particles with toroidal and rod-like morphologies. However, it has not been shown to what degree kinetic factors (e.g., condensate nucleation) versus thermodynamic factors (e.g., DNA bending energy) determine experimentally observed relative populations of toroids and rods. It is also not clear how multimolecular DNA toroids and rods interconvert in solution. We have conducted a series of condensation studies in which DNA condensate morphology statistics were measured as a function of time and DNA structure. Here, we show that in a typical in vitro DNA condensation reaction, the relative rod population 2 min after the initiation of condensation is substantially greater than that measured after morphological equilibrium is reached (ca. 20 min). This higher population of rods at earlier time points is consistent with theoretical studies that have suggested a favorable kinetic pathway for rod nucleation. By using static DNA loops to alter the kinetics and thermodynamics of condensation, we further demonstrate that reported increases in rod populations associated with decreasing DNA length are primarily due to a change in the thermodynamics of DNA condensation, rather than a change in the kinetics of condensate nucleation or growth. The results presented also reveal that the redistribution of DNA from rods to toroids is mediated through the exchange of DNA strands with solution.     

Cationic silanes stabilize intermediates in DNA condensation.

In vitro condensation of DNA has been widely studied to gain insight into the mechanisms of DNA compaction in biological systems such as chromosomes and phage heads and has been used to produce nanostructured particles with novel material and functional properties. Here we report on the condensation of DNA in aqueous solutions by cationic silanes, which combine the condensing properties of polyamines with the cross-linking chemistry of silanes. DNA can be reversibly condensed into classical toroidal and rod-shaped structures with these agents. At low silane concentrations DNA forms a variety of looped structures with well-defined characteristics, including flower- and sausage-shaped forms. These structures suggest that at low silane concentrations a DNA-DNA contact in which the strands are at very large angles to each other is stabilized. Changes in these structures observed as a function of silane concentration suggest possible pathways for the formation of toroids and rods.     

DNA toroids: stages in condensation.

The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.     

Bacterial protein HU dictates the morphology of DNA condensates produced by crowding agents and polyamines

Controlling the size and shape of DNA condensates is important in vivo and for the improvement of nonviral gene delivery. Here, we demonstrate that the morphology of DNA condensates, formed under a variety of conditions, is shifted completely from toroids to rods if the bacterial protein HU is present during condensation. HU is a non-sequence-specific DNA binding protein that sharply bends DNA, but alone does not condense DNA into densely packed particles. Less than one HU dimer per 225 bp of DNA is sufficient to completely control condensate morphology when DNA is condensed by spermidine. We propose that rods are favored in the presence of HU because rods contain sharply bent DNA, whereas toroids contain only smoothly bent DNA. The results presented illustrate the utility of naturally derived proteins for controlling the shape of DNA condensates formed in vitro. HU is a highly conserved protein in bacteria that is implicated in the compaction and shaping of nucleoid structure. However, the exact role of HU in chromosome compaction is not well understood. Our demonstration that HU governs DNA condensation in vitro also suggests a mechanism by which HU could act as an architectural protein for bacterial chromosome compaction and organization in vivo.     

Atomic force microscopy analysis of intermediates in cobalt hexammine-induced DNA condensation

The packaging pathway of cobalt hexammine-induced DNA condensation on the surface of mica was examined by varying the concentration of Co(NH3)6(3+) in a dilute DNA solution and visualizing the condensates by atomic force microscopy (AFM). Images reveal that cobalt hexammine-induced DNA condensation on mica involves well-defined structures. At 30 microM Co(NH3)6(3+), prolate ellipsoid condensates composed of relatively shorter rods with linkages between them are formed. At 80 microM Co(NH3)6(3+), the condensed features include toroids with average diameter of approximately 240 nm as well as U-shaped and rod-like condensates with nodular appearances. The results imply that the condensates, whether toroids, U-shaped or rod-like structures have similar intermediate state which includes relatively shorter rod-like segments. The average size of the condensed toroids after incubated at room temperature for 5 h (approximately 240 nm) is much larger than that incubated for 0.5 h (approximately 100 nm). The results indicate that the condensation of DNA by Co(NH3)6(3+) is a kinetic-controlled process.     

Entrapment and condensation of DNA in neutral reverse micelles

DNA condensation and compaction is induced by a variety of condensing agents such as polycations. The present study analyzed the structure of plasmid DNA (DNA) in the small inner space of reverse micelles formed from nonionic surfactants (isotropic phase). Spectroscopic studies indicated that DNA was dissolved in an organic solvent in the presence of a neutral detergent. Fluorescent quenching of ethidium bromide and of rhodamine covalently attached to DNA suggested that the DNA within neutral, reverse micelles was condensed. Circular dichroism indicated that the DNA structure was C form (member of B family) and not the dehydrated A form. Concordantly, NMR experiments indicated that the reverse micelles contained a pool of free water, even at a ratio of water to surfactant (Wo) of 3.75. Electron microscopic analysis also indicated that the DNA was in a ring-like structure, probably toroids. Atomic force microscopic images also revealed small, compact particles after the condensed DNA structures were preserved using an innovative cross-linking strategy. In the lamellar phase, the DNA was configured in long strands that were 20 nm in diameter. Interestingly, such DNA structures, reminiscent of "nanowires," have apparently not been previously observed.