氯仿—异戊醇—核糖核酸酶法—快速提取植物DNA的好方法

近几年,我们在外源DNA导入小麦研究工作中,采用氯仿-异戊醇-核糖核酸酶法提取DNA,得到了比较满意的结果.现将该方法介绍如下.材料和方法材料:大麦、小麦、野燕麦等.取其种苗(10-15天苗龄)或孕穗期的幼穗、嫩茎、嫩叶等.     

火球菌8-氧鸟嘌呤DNA糖苷酶基因克隆、表达纯化和酶学性质研究

【目的】在大肠杆菌中表达火球菌8-氧鸟嘌呤DNA糖苷酶,纯化得到重组火球菌8-氧鸟嘌呤DNA糖苷酶,在此基础上系统研究火球菌8-氧鸟嘌呤DNA糖苷酶的酶学特征。【方法】构建8-氧鸟嘌呤DNA糖苷酶重组表达质粒,将重组质粒转化Escherichia coli Rosetta(DE3),利用IPTG诱导表达重组蛋白,通过Ni2+亲和层析柱纯化重组蛋白;最后利用含8-氧鸟嘌呤损伤的寡核苷酸作为底物,测定8-氧鸟嘌呤DNA糖苷酶的酶学性质。【结果】在大肠杆菌中成功诱导表达了重组火球菌8-氧鸟嘌呤DNA糖苷酶,经Ni2+亲和纯化后蛋白纯度大于95%。在体外鉴定了重组火球菌8-氧鸟嘌呤DNA糖苷酶的酶学性质。结果表明重组火球菌8-氧鸟嘌呤DNA糖苷酶可以切除DNA中的8-氧鸟嘌呤(8-Oxo-G,GO)损伤碱基,并且具有AP裂解酶活性。重组火球菌8-氧鸟嘌呤DNA糖苷酶催化反应的最适pH值和温度分别是pH 8.5和55°C。除Zn2+对火球菌8-氧鸟嘌呤DNA糖苷酶的酶促反应有明显的抑制作用外,实验中测定的其它二价离子(Mn2+,Mg2+,Ca2+,Ni2+,Co2+,Cu2+)对其没有明显的影响。离子强度在50-100 mmol/L范围内对其酶促反应影响不大,超过100 mmol/L时有明显的抑制作用。与8-氧鸟嘌呤互补的碱基差异对火球菌8-氧鸟嘌呤DNA糖苷酶切除8-氧鸟嘌呤损伤的效率影响不大;但与单链DNA相比,双链DNA是优选底物,切割效率如下:GO/C≈GO/G≈GO/T≈GO/AGO/-。【结论】在大肠杆菌中成功表达,并Ni2+亲和纯化了火球菌8-氧鸟嘌呤DNA糖苷酶,生化研究表明制备的重组蛋白具有8-氧鸟嘌呤DNA糖苷酶活性,可能负责切除火球菌基因组DNA中的8-氧鸟嘌呤损伤。     

DNA芯片在0-1规划问题中的应用

生物芯片技术和DNA计算分别是近年来生命科学与信息科学的新兴研究领域,对信息高度并行的获取与处理是二者的本质特性.而0-1规划问题作为运筹学中一个重要的问题,到目前为止还没有好的算法.在DNA计算和DNA芯片基础上,提出了基于DNA芯片解决0-1规划问题的DNA计算新模型,与以往DNA计算模型相比,该模型具有高信息量和操作易自动化的优点.同时指出DNA芯片技术有望作为新型生物计算的芯片.     

多粘类芽孢杆菌SC2 xynD和gluB的克隆及序列分析

β-1,4-木聚糖酶和β-1,3-1,4-葡聚糖酶活性检测结果表明,多粘类芽孢杆菌(Paenibacillus polymyxa)SC2能同时产生β-1,4-木聚糖酶和β-1,3-1,4-葡聚糖酶.以菌株SC2的基因组DNA为模板,通过TAIL-PCK方法克隆到4 807bp的DNA片段.DNAMAN软件分析,该DNA片段包含2个开放阅读框(ORF),ORF1长度为1 908 bp,编码含635个氨基酸、分子量为67.8kD的蛋白;ORF2长度为714 bp,编码含237个氨基酸、分子量为26.8kD的蛋白.BLAST分析结果表明,ORFI与已报道的P.polymyxa ATCC 842的xynD基因相似性为94%.ORF2与P.polymyxaWY110的gluB基因相似性为99%.基因序列的系统发育分析进一步说明.ORFI和ORF2分别为β-1,4-木聚糖酶基因xynD和β-1,3-1,4-葡聚糖酶基因gluB.     

14-3-3θ基因过表达载体的构建及其过表达乳腺癌细胞株的建立

目的:为进一步研究14-3-3θ基因对乳腺癌生物学行为的影响,构建14-3-3θ基因真核细胞过表达重组载体并建立其过表达的乳腺癌细胞株。方法:合成的14-3-3θ基因重组在p ENTR 3C DUAL载体中,课题组采用DNA重组技术将14-3-3θ基因剪切分离后,插入真核表达质粒载体pc DNA4.0中,重组得到真核过表达载体pc DNA4.0-14-3-3θ,并用其转染293T细胞后用western blot进行验证,将经过验证的真核过表达载体pc DNA4.0-14-3-3θ转染乳腺癌细胞后用zeocin筛选,筛选出的乳腺癌细胞经过western-blot和PCR验证确为稳定过表达14-3-3θ的乳腺癌细胞。结果:成功构建14-3-3θ基因过表达真核载体并建立其过表达乳腺癌细胞株,揭示14-3-3θ基因可能在乳腺癌的早期诊断和预后预测中具有重要作用。     

合成酮内酯类3-脱氧-3-羰基-红霉内酯B糖多孢红霉菌M的构建

大环内酯类抗生素基因工程是近年来研究的一个新领域,迄今已合成了100多种新的聚酮类化合物。以糖多孢红霉菌A226基因组DNA为模板,用重叠PCR方法扩增出去除KR6酶域DNA的约32kb DNA片段,克隆于pWHM3载体,构建了同源重组质粒pWHM2201。PEG介导原生质体转化法将pWHM2201转入糖多孢红霉菌A226,并整合于染色体红霉素合成基因位点。整合体在R3M斜面上生长两代后,制备原生质体涂R3M平皿。利用PCR鉴定筛选出8株KR6敲除的突变体糖多孢红霉菌M(1-8)。ZabsPec Fab质谱鉴定,证实糖多孢红霉菌M1合成了3-脱氧-3-羰基-红霉内酯B,一种新的酮内酯类化合物。 〖HJ0     

温度对UV-B诱导的烟草叶圆片DNA损伤的影响

环丁烷嘧啶二聚体(CPD)和6-4光产物(6-4PP)是两种主要的UV-B诱导的DNA光损伤产物。利用单克隆抗体酶联免疫吸附分析法(ELISA),研究了温度对UV-B诱导的烟草叶圆片DNA损伤的影响。室温(24℃)条件下,UV-B处理引起了烟草叶圆片DNA中CPD和6-4PP的积累。0℃条件下,UV-B处理的烟草叶圆片DNA中CPD和6-4PP的积累比室温下分别降低了9.8%和12%。UV-B诱导的DNA损伤曾被认为是纯粹的光化学过程而与不受温度影响,而本实验结果表明,UV-B诱导的烟草叶圆片DNA形成CPD和6-4PP的过程具有温度依赖性。这一特性有利于植物对全球变化的适应,因而具有重要的生态学意义。     

DNA稳定的银纳米簇诱导的双酶催化转化检测研究

为了实现对碱性磷酸酶和邻-磷酸-L-酪氨酸的同时检测,本研究利用 DNA 稳定的银纳米簇（AgNCs／DNA）诱导的生物催化转化,基于醌类物质对 DNA 稳定的银纳米簇的荧光的猝灭效应,通过酪氨酸酶能与酪胺、多巴胺以及酪氨酸反应生成醌类物质,间接地达到了对酪胺、多巴胺以及酪氨酸的定量检测。AgNCs／DNA 还被进一步用于双酶催化级联反应,通过碱性磷酸氧化酶（ALP）和酪氨酸酶（TYR）组成的双酶系统,实现了对碱性磷酸酶和邻-磷酸-L-酪氨酸的高灵敏度检测,检测限分别达到2．5×10－5μmol·L－1和0．01μmol·L－1。     

10-23型DNA酶作为鉴定mRNA靶点有效性的新工具

10-23DNA酶是能主动切割mRNA的一类反义寡核苷酸.利用10-23DNA酶的直接切割作用验证mRNA结构靶点的有效性.对筛选的绿色荧光蛋白(GFP)基因mRNA的4个靶点平行设计了4条反义寡核苷酸和4条10-23DNA酶,对照组反义寡核苷酸将最佳靶点——靶点2的反义寡核苷酸突变2个碱基,对照组10-23DNA酶将靶点2的10-23DNA酶结合臂中央突变2个碱基.体外4条10-23DNA酶切割mRNA的结果和相应的4条反义寡核苷酸依赖的RNaseH降解结果完全相似,细胞内4条10-23DNA酶对绿色荧光蛋白的表达抑制作用与相应的4条反义寡核苷酸相似,表明10-23DNA酶显示的最佳作用靶点同样是最佳作用效果的反义寡核苷酸结合靶.10-23DNA酶可以作为评价mRNA结构靶点有效性的新工具.     

电离辐射诱导DNA—PKcs缺失小鼠T细胞特异的V（D）J重组恢复

NA依赖的蛋白激酶 (DNA PK)是一种DNA活化的核丝氨酸苏氨酸蛋白激酶。DNA PK由一种与DNA末端结合的调节亚单位异构二聚体Ku蛋白和DNA PK催化亚单位 (DNA PKcs)组成。DNA PK在DNA暴露于电离辐射后诱导的双链损伤修复中起主要作用。为了更好地了解与DNA PKcs缺失相关的DNA修复缺陷的本质。建立了DNA PKcs-/ -小鼠胚胎成纤维细胞株和裸鼠模型 ,调查这些突变的细胞和小鼠对DNA损害的反应。DNA PKcs-/ -细胞对电离辐射超敏感 ,在克隆形成实验中显示较低的生成率。同样 ,DNA PKcs-/ -小鼠也显示极大的放射敏感性 ,新生DNA PKcs-/ -小鼠用亚致死剂量电离辐射处理恢复T细胞受体 (TCR) β重组和T细胞成熟。然而 ,放射辐射并不恢复B细胞发育。DNA PKcs-/ -小鼠最终发生胸腺淋巴瘤。这些结果提示DNA双链断裂 (DSB)修复 ,V(D)J重组和淋巴瘤发生之间的相互关系。提供一种体内模型以阐明DNADSB修复调节、V(D)J重组和淋巴瘤发生分子机制三者之间的关键通路     

The Replication of DNA: II. The Number of Polynucleotide Strands in the Conserved Unit of DNA

The kinetics of degradation of DNA by deoxyribonuclease II have been studied, using the techniques of light scattering, viscosity, and titration. Theoretical equations have been derived for both random and non-random attacks, and all assumptions have been evaluated. It has been shown that these equations permit a valid calculation of the number of polynucleotide strands per molecule. The results have been verified by two independent experimental methods. DNA from proliferating sources was found to be four-stranded; DNA from non-proliferating sources was found to be two-stranded. The implications of these findings are discussed.     

Multimerization-cyclization of DNA fragments as a method of conformational analysis

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.     

Kinetics of adsorption of extended ligands on DNA at small fillings

In the present work, the adsorption kinetics of extended ligands on DNA duplexes at small fillings when molecules of DNA duplexes are on the underlayer within diffusion layer has been investigated. Both diffusion of ligands in solution (diffusion stage) and adsorption of ligands (kinetic stage) are taken into consideration at adsorption of ligands on DNA duplexes. Nonlinear system of differential equations describing adsorption of ligands where not only diffusion stage but also kinetic stage is taken into account, is obtained, moreover the equations allow localizing duplexes in arbitrary place within diffusion layer. Numeric solution of the equations makes possible to investigate the filling kinetics of DNA duplexes by ligands depending on parameters controlling adsorption process. It has been shown that depending on relation between adsorption parameters different kinetic regimes of adsorption – kinetic, complex, and diffusion regimes may be realized.     

Randomly distributed DNA double-strand breaks as measured by pulsed field gel electrophoresis: A series of explanatory calculations

The aim of this article is to characterize expressions of relevance to the interpretation of pulsed field gel electrophoresis (PFGE) experiments where randomly distributed double-strand breaks (DSBs) are detected as smears of DNA fragments. Specifically, equations for conversion of percentages of fragments in defined size ranges to DSBs were derived. Several models have been used, one of which is based on theoretically fragmented DNA from the fission yeastSchizosaccharomyces pombe, which has three PFGE separable chromosomes.     

A simple method for the determination of DNA molecular weight distribution

A method is described for the molecular weight distribution of DNA which is determined from sedimentation-velocity analysis. Knowing the distribution of sedimentation coefficients for a single DNA concentration it is possible to extrapolate such a distribution to infinite dilution of the solute in a simple way. Two versions (using two or three terms of a series) of extrapolating equations are considered and discussed in detail. The sedimentation coefficients distribution calculated from these equations differs only insignificantly with that obtained in a conventional way.     

Method for determining the extent and copy number of overlapping and nonoverlapping segments of integrated viral genomes.

We analyzed the method of exhaustive hybridization of single-stranded DNA and derived a general relationship between the fraction of the probe DNA hybridized and the sizes and copy numbers of the segments of the viral genome integrated in cellular DNA. The equations employed can be used to analyze integrated DNA comprised of overlapping and nonoverlapping segments of the viral genome. Using these equations, we analyzd the adenovirus type 2 DNA content of a series of hamster cell lines transformed by adenovirus type 2 and several adenovirus type 2-simian virus 40 hybrid viruses. We found no eividence that the integrated viral DNA is comprised of overlapsping segments. However, the number of copies of the integrated segments varies between lines cloned from the same transformed isolate, and copy numbers change during in vivo passage of transformed cells.     

Studies on the toxicity and binding kinetics of abrin in normal and Epstein-Barr virus-transformed lymphocyte culture-I. Experimental results - 4

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein-Barr virus (EBV)-transformed lymphocytes have been previously investigated. Using data on EBV-transformed lymphocyte cell density as a function of both time and dose of abrin, the authors introduced the concept of self- and cross-coupling metabolic variables as a means of understanding how abrin affected DNA and protein uptake. In this paper, the self-coupling constant is studied in more detail and the relationship between DNA and protein synthesis is further expanded. We find that there is a significant linear relationship between DNA and protein synthesis in normal lymphocyte culture as measured by abrin interaction in the culture. We further find that there is a much stronger relationship between these variables in EBV-transformed lymphocyte culture. This relationship is further examined, and possible analytic equations are expressed.     

Theoretical analysis of gel electrophoretic data for interaction of lysine rich histone with supercoiled DNA.

We demonstrate the possibility of using gel electrophoresis as a technique for the quantitative analysis of interaction of lysine rich histone with DNA. On the basis of theoretical framework for extended ligand binding to one-dimensional lattices such as DNA we have set up systems of equations which relate the ligand-to-DNA ratio to the observed gel migration distance of the complex. From the analysis of experimental data for gel electrophoresis of supercoiled DNA in the presence of lysine rich histones we have found that the observed variation of electrophoretic mobilities of the histone-DNA complexes at low histone-to-DNA ratios can be described by a non-cooperative binding behaviour. At this limit we have estimated the intrinsic binding constant to be of the order of 10(3) M-1.     

Analysis of the kinetic hairpin transfer model for parvoviral DNA replication

All linear DNA molecules face special problems in replicating their 5' ends, as DNA polymerases add nucleotides only to pre-existing strands with free 3'-OH groups. Parvoviruses, a group of small animal viruses with a linear single-stranded DNA genome, cope with this problem by having palindromic terminal sequences that can fold back on themselves to form hairpin structures essential in priming DNA replication. The 3' terminal sequence that initiates replication becomes reversed in orientation during the process, and if the palindrome is imperfect, two different, reverse-complementary terminal sequences are generated. The relative abundances of the terminal sequence orientations at each end of the DNA molecules can be measured and give information about the replication process. From such clues, we developed a "kinetic hairpin transfer model" based on differential rates of hairpin formation and inversion processes depending on the conformations of the 3' termini. Numerical studies showed that this simple idea can account for the diverse pattern of DNA distributions observed in the family Parvoviridae. In this paper, we simplify the model to a set of coupled linear first-order ordinary differential equations in order to delineate its essential properties by Perron-Frobenius theory. Secondly, we examine our assumption of linear kinetics by modeling enzyme catalysis of the component steps of the hairpin transfer process. We show that the rate-determining step of the process is the binding of initiation complex to the self-priming hairpin structures. Furthermore, we find that if the replication machinery is saturated by DNA substrate late in an infection, the differential equations become non-linear but the steady-state DNA distribution is still given by the solution of our original linear equations.