DNA改组(DNA shuffling)及其研究进展

自1994年首次提出至今,DNA改组已经成为定向进化最为有效的方法之一.无论DNA、蛋白质还是生物体的进化,DNA改组都有十分突出的表现.通过对十余年来DNA改组的发展作以简要综述,为日后相关研究的开展提供理论基础.     

DNA简史(续)

1955美国生物化学家、西班牙出生的奥乔亚(Severo Ochoa)和其同事们合成了一种类似RNA 的分子。在研究细菌的过程中,他们发现了多核苷酸磷酸化酶。当把这种酶加到含有核苷二磷酸盐、缓冲盐和镁离子的介质中时,就能制出人造的 RNA。     

Comparison Between poly(dG-dC)·poly(dG-dC) and DNA Modified by cis-diamminedichloroplatinum (II): Immunological and Spectroscopic Studies

Abstract

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG)·poly(dC) is larger than to poly (dG-dC)·poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC) ·poly(dG-dC), poly(dA-dC) ·poly(dG-dT) and poly(dA-dG)·poly(dC-dT). In the competition between poly(dG-dC) ·poly (dG-dC) (B conformation) and poly(dG-br⁵dC) ·poly(dG-br⁵dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)_n·(GC)_nsequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized by the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-m⁵dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diammine- dichloroplatinum(II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m⁵dC) in the Z conformation. When they bind to poly(dG-m⁵dC) in the B conformation, the conformations of poly(dG-m⁵dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.     

Charge Transport in Poly(dG)–Poly(dC) and Poly(dA)–Poly(dT) DNA Polymers

We investigate the charge transport in synthetic DNA polymers built up from single type of base pairs. In the context of a polaronlike model, for which an electronic tight-binding system and bond vibrations of the double helix are coupled, we present estimates for the electron-vibration coupling strengths utilizing a quantum-chemical procedure. Subsequent studies concerning the mobility of polaron solutions, representing the state of a localized charge in unison with its associated helix deformation, show that the system for poly(dG)–poly(dC) and poly(dA)–poly(dT) DNA polymers, respectively possess quantitatively distinct transport properties. While the former supports unidirectionally moving electron breathers attributed to highly efficient long-range conductivity, the breather mobility in the latter case is comparatively restrained, inhibiting charge transport. Our results are in agreement with recent experimental results demonstrating that poly(dG)–poly(dC) DNA molecules acts as a semiconducting nanowire and exhibit better conductance than poly(dA)–poly(dT) ones.     

重组DNA技术(续)

四、DNA的体外重组 DNA重组技术,当用于干扰素、胰岛素、疫苗、免疫球蛋白等物质的生产时,目前多半采用从mRNA反转录成cDNA,然后和载体DNA重组,再转化至大肠杆菌内进行复制和表达。在某一基因结构已清楚的情况下,亦可人工合成该基因(如人胰岛素基因等),再将该基因嵌入载体引进大肠杆菌中。若从基因库中分离出的基因,由于已含有内含子(Intron),     

Preparation and characterization of chitosan and trimethyl-chitosanmodified poly-(ε-caprolactone) nanoparticles as DNA carriers

The purpose of this research was to prepare poly-(ε-caprolactone) (PCL) particles by an emulsion-diffusion-evaporation method using a blend of poly-(vinyl alcohol) and chitosan derivatives as stabilizers. The chitosan derivatives used were chitosan hydrochloride and trimethyl chitosans (TMC) with varying degrees of quaternization. Particle characteristics-size, zeta potential, surface morphology, cytotoxicity, and transfection efficiency-were investigated. The developed method yields PCL nanoparticles in the size range of 250 to 300 nm with a positive surface charge (2.5 to 6.8 mV). The cytotoxicity was found to be moderate and virtually independent of the stabilizers' concentration with the exception of the highly quaternized TMC (degree of substitution 66%) being significantly more toxic. In immobilization experiments with gel electrophoresis, it could be shown that these cationic nanoparticles (NP) form stable complexes with DNA at a NP:DNA ratio of 3:1. These nanoplexes showed a significantly higher transfection efficiency on COS-1 cells than naked DNA. Published: August 10, 2005     

一个简便的DNA改组(DNA shuffling)操作程序

介绍了一个简便的DNA改组操作程序.首先利用PCR扩增了两段具有高度序列同源性的1 700 bp左右的基因片段,两者相比较同源性大于93%.然后将其等量混合后,在Mg²⁺存在的条件下,用DNaseⅠ切割成10～50 bp的小片段.这些小片段在不外加引物的前提下,利用PCR反应进行重聚,再将重聚物经过两轮正常的PCR扩增,获得了与原来片段大小相当的基因片段.这一技术有利于从一组序列同源性程度较高的基因库构建随机嵌合基因.     

Comparison of Au(III) and Ga(III) Ions’ Binding to Calf Thymus DNA: Spectroscopic Characterization and Thermal Analysis

Metals have been studied as potential chemotherapeutic agents for cancer therapies due to their high reactivity toward a wide variety of substances. The characterization of metal ion-binding capacities is essential to understand the possible effects of metals on target biomolecules. In the present study, biochemical effects of Au(III) and Ga(III) ions on calf thymus DNA (ctDNA) were studied comparatively via bioanalytical, spectroscopic, and thermal methods. Briefly, UV-Vis absorbance spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy were utilized for spectroscopic characterization, and isothermal titration calorimetry (ITC) measurements were performed for thermal analysis. Our results reveal that both Au(III) and Ga(III) ions are capable of interacting with ctDNA, and Au(III) ions display a more favorable interaction and a higher binding affinity. ITC analyses indicate that the Au(III)-DNA interaction displays a binding affinity (K_a) around 1.43?×?10⁶ M^?1, while a K_a around 1.17?×?10⁵ M^?1 was observed for the Ga(III)-DNA binding. It was suggested that both metal ions are unlikely to change the structural B-conformation while interacting with ctDNA.     

DNA与个体发育调控(2)

生物结构的形成需要各种细胞按照类型分别聚集,这主要是通过细胞表面的钙黏蛋白实现的。形成片、管、腔等结构需要细胞具有极性;上皮表面上的的结构如纤毛、羽毛、鳞片、毛发具有方向性,也需要有关细胞具有极性。细胞的极性是由细胞内和细胞表面的一些蛋白质聚合物彼此拮抗并不对称分布而形成的。细胞之间通过Notch蛋白及其配体之间的相互作用导致彼此相邻的细胞向不同的分化方向发展。这些"成型分子"在胚胎发育过程中都发挥重要的作用。     

DNA与个体发育调控(1)

多细胞生物的身体都是由一个细胞发育而来的,因此该细胞内的遗传物质DNA应携带有建造身体结构的全部信息指令。然而,在DNA序列中却只有为基因编码的序列,以及控制基因表达时间、地点、多少的调控序列,并没有如何建造身体的具体信息指令。DNA的蓝图作用,是通过各种蛋白质分子的顺序表达来形成各种类型的细胞,同时通过一些细胞分泌的信息分子指挥周围细胞表达出能够产生机械力的蛋白质分子,让细胞按照特异方式彼此结合,从而实现个体发育的精准调控。     

DNA与个体发育调控(4)

控制细胞命运的信息分子可以在细胞之间扩散,在较大程度上影响众多细胞的命运,是较高层次的指挥。但要形成各种具体的生物结构,例如昆虫的复眼、翅膀、触角和腿,还需要具体的相关基因完成这项工作。Hox家族和Pax家族的基因就专门负责控制此类相关基因,它们能够动员起为建造一个具体器官所需要的全部基因来完成器官的建造工作。这些基因的历史非常久远,从简单的多细胞动物到人都使用这些基因。     

Chromosome numbers and DNA?C values in the genus Lippia (Verbenaceae)

The genus Lippia comprises herbs, shrubs, and small trees, including many species with medicinal properties. The species are distributed throughout South and Central America and Tropical Africa, but the majority of them occur in Brazil, Paraguay, and Argentina. The DNA?C value of 28 Brazilian species has been estimated by flow cytometry. Estimated DNA?C values ranged from 0.825?pg (L. corymbosa) to 2.150?pg (L.?brasiliensis). In addition, new chromosome numbers of 12 species have also been described, and meiotic cells with 12, 13, and 14 chromosome pairs were observed. A straightforward correlation between chromosome number and DNA?C value was not observed, probably due to two outlier species of Lippia that have been transferred from the genus Lantana. In general, the data confirm previous reports regarding the variation within the taxonomic sections and also suggest a new revision in section Zapania. Aspects of karyotypic evolution of the genus are also discussed.     

Interpretation of DNA vibration modes: I-The guanosine and cytidine residues involved in poly(dG-dC)·poly(dG-dC) and d(CG)3·d(CG)3

Abstract

A normal coordinate analysis has been carried out on guanosine and cytidine residues appearing in oligo and polynucleotides by using a simplified valence force field that allows the vibrational spectra of 5′-dGMP and 2′-deoxycytidine molecules to be reproduced. The role of both C2′-endo and C3′-endo conformations on sugar pucker, as well as that of glycosidic torsion angle (χ), on several characteristic vibration modes of these residues have been studied. The present calculations based on a non-redundant set of internal coordinates preserving the harmonic approximation of the potential field, allows us to explain quite satisfactorily the modifications of the vibrational spectra in the 1550-1250 cm^?1 and 785-500 cm^?1 regions, when the right → left-handed conformational transition occurs.     

DNA与个体发育调控(3)

在生物体形成的过程中,不仅细胞-细胞之间的直接接触起重要作用,例如细胞分类聚集、形成片状和管状结构、相邻细胞各向不同的类型发展等,而且控制中心的细胞还会分泌出指导周围细胞生长分化的信息分子。这些信息分子能够在细胞之间移动,在比较大的尺度上影响众多细胞的命运。这些信息分子与受控细胞上的受体结合,在细胞中启动信号传递链,使这些细胞向特定的方向发展。     

DNA–DNA Hybridization Evidence for the Recent Origin of Marmots and Ground Squirrels (Rodentia: Sciuridae)

A molecular analysis, based on DNA/DNA hybridization experiments, examined seven species of the sciurid tribe Marmotini, in order to evaluate their evolutionary relationships. The branching pattern obtained from a neighbor-joining analysis and least-squares methods indicates that spermophiles and marmots are closely related. This result is in agreement with previous studies based on biochemical data which have suggested a Miospermophilus origin of Marmota (like Spermophilus), instead of the Protospermophilus origin usually proposed by paleontology. Using a molecular time scale calibrated by the fossil record of Sciurus (used here as an outgroup), we estimate the marmot/ground squirrel divergence at 6 My, at the same time as the major lineages of ground squirrels diverged from each other. Consequently, the species of Marmota appear as one of the numerous specialized forms derived from the explosive radiation of the Spermophilus lineage in the late Miocene.     

Interactions Between Esterified Whey Proteins (α-Lactalbumin and β-Lactoglobulin) and DNA Studied by Differential Spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated β-lactoglobulin and of methylated α-lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated β-casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Structure?Activity Relationship of Polypyridyl Ruthenium(II) Complexes as DNA Intercalators,DNA Photocleavage Reagents,and DNA Topoisomerase and RNA Polymerase Inhibitors

To investigate the relationship between the molecular structure and biological activity of polypyridyl Ru^II complexes, such as DNA binding, photocleavage ability, and DNA topoisomerase and RNA polymerase inhibition, six new [Ru(bpy)₂(dppz)]²⁺ (bpy=2,2′‐bipyridine; dppz=dipyrido[3,2‐a:2,′,3′‐c]phenazine) analogs have been synthesized and characterized by means of ¹H‐NMR spectroscopy, mass spectrometry, and elemental analysis. Interestingly, the biological properties of these complexes have been identified to be quite different via a series of experimental methods, such as spectral titration, DNA thermal denaturation, viscosity, and gel electrophoresis. To explain the experimental regularity and reveal the underlying mechanism of biological activity, the properties of energy levels and population of frontier molecular orbitals and excited‐state transitions of these complexes have been studied by density‐functional theory (DFT) and time‐depended DFT (TDDFT) calculations. The results suggest that DNA intercalative ligands with better planarity, greater hydrophobicity, and less steric hindrance are beneficial to the DNA intercalation and enzymatic inhibition of their complexes.     

DNA重组技术(Ⅰ) 小量快速提取质粒DNA方法

质粒DNA的提取是最基础的DNA重组技术。国内外已经发展了许多方法用于批量提取质粒DNA。这些方法都可以获得满意的效果。这里介绍两种小量快速提取质粒DNA的方法,可用于重组子筛选及酶切分析。 (一)碱裂解法 1.将过夜培养的菌液1.5ml转移到小离心管内,15.000r/min离心30sec,弃去上清,将离心管倒置尽量除去培养液。 2.将沉淀的菌体再悬于100ml冰冷的溶液A中(50mM葡萄糖,25mM Tris pH8.0,10mM EDTA),不必要加入溶菌酶。 3.向A液中加入新配制的溶液B200μl(0.2N NaOH,1％SDS),迅速倒置几次,混合两溶液,放在冰浴上3—5分钟。 4.再加入150μl溶液C(5M醋酸钠60ml,冰醋酸11.5ml,蒸馏水28.5ml)混均后冰浴3～5分钟。