An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.     

Thermostability of subtilisin nattokinase obtained by site-directed mutagenesis

To study the thermostability of Nattokinase(subtilisin NAT,NK),three double mutant plasmids(pET-28a-NKG61C/S98C,pET-28a-NKT22C/S87C,pET-28a-NKS24C/S87C)were constructed by site-directed mutagenesis.Target enzymes were detected using SDS-PAGE and disulfide bond formation was detected using Western blotting analysis.Thermostability was tested by rates of inactivation at certain temperature.The results showed that disulfide bond was not formed within two cysteines and the thermostability of three double mutants was not increased compared with the wild-type NK.The thermostability of NK performed in Ca2+was stronger than in ethylenediaminetetraacetic acid(EDTA).But when the temperature reached 62℃,the enzymes rapidly denatured and inactivated even in the presence of Ca2+.Although the thermostability of mutants was not increased,this study shows a tendency of improving thermostability of NK in protein engineering.     

计算机辅助木糖还原酶定点突变的生物信息学分析

木糖还原酶（xylose reductase,XR）是木糖代谢生成乙醇途径中一个重要的酶,目前利用纤维素生成酒精的关键问题之一：木糖代谢过程中XR和木糖醇脱氢酶（xylitol dehydrogenase,XDH）的氧化还原不平衡。本研究借助生物信息学手段（酶三维结构建模、酶和辅酶分子对接）,充分分析数据库资源,找到了一些可能影响XR酶活性或辅酶依赖性的关键氨基酸。毕赤氏酵母XR与NADP之间有Lys21（K）、Val222（V）、Glu223（E）、Phe236（F）和Thr273（T）;毕赤氏酵母XR与NAD之间有Val222（V）、Glu223（E）、Phe236（F）、Glu237（E）和Thr273（T）;热带假丝酵母XR与NADP之间有Asn278（N）和Arg282（R）。对比两种辅酶与毕赤氏酵母XR形成氢键的氨基酸,如果使毕赤氏酵母XR只与辅酶NAD结合,则可以将Lys21替换成其它的氨基酸,因Lys21在所有XR序列中完全保守,需要进行氨基酸替代模拟计算预实验,在确保酶三维结构不变及NAD可以结合XR的前提条件下替代Lys21;如果使毕赤氏酵母XR只与辅酶NADP结合,则可以将Glu237（不完全保守）替换成其它的氨基酸。另外,还可以根据需要将这些形成氢键的氨基酸进行组合替代。要改变热带假丝酵母XR的NADP依赖性,可以替代Asn278（N）和/或Arg282（R）（不完全保守）。本研究为进一步酶的理性设计（提高活性及改变辅酶依赖性）并在分子水平上对木糖还原酶进行改造打下了基础。     

Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis

Nuclear magnetic resonance (NMR) studies have shown that two distinct folded conformations of staphylococcal nuclease coexist in solution and that these two states can interconvert directly without passing through an unfolded state. These experiments have also revealed that the two forms have very different folding kinetics, although the possibility that one component is an obligatory intermediate for the folding of the other form could be discounted. Here we report NMR data which show that alternative unfolded states are also distinguishable. These observations led us to hypothesize that cis/trans isomerism at a single peptide bond between a proline and its preceding residue might be the origin of the conformational multiplicity. Proline 117 was identified as a likely candidate for the site concerned and a mutant protein, in which Pro 117 was replaced by Gly, was constructed in order to test this. Alternative conformations are not observed in the spectrum of this mutant, lending powerful support to this hypothesis.     

Molecular dynamics simulation of site-directed mutagenesis of HIV-1 Tattrans-activator

The Cys-rich domain, core region and basic domain are highly conserved and very important to thetrans-activation activity of HIV-1 Tattrans-activator. The three-dimensional structures of 6 mutants of HIV-1 Tat protein were constructed with the methods of molecular dynamics simulation. The variations of the structures of the mutants have been analyzed and the factors that led to abolishment oftrans-activation activity have been discussed.     

Location of functional regions of acetylcholine receptor alpha-subunit by site-directed mutagenesis

The availability of cloned cDNAs encoding the four subunits of the Torpedo acetylcholine receptor, which can be expressed to make functional receptors in Xenopus oocytes, has made possible a detailed investigation of the functions of the different structural components of the receptor. The functional analysis of receptors with alpha-subunits altered at specific sites by site-directed mutagenesis of the cDNA has allowed the location of specific regions of the alpha-subunit molecule involved in acetylcholine binding and forming a transmembrane ionic channel.     

Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis

The ability of polyoma virus to transform cells results primarily from the action of one of the virus-coded early proteins, called middle-T antigen. Middle-T has an associated tyrosine-specific protein kinase activity that can be measured in vitro and results in the phosphorylation of middle-T itself. Almost all mutants so far tested that lack the ability to transform cells, also lack associated kinase activity. Attempts to map within middle-T the tyrosine residue(s) that are phosphorylated in vitro suggest that a likely site of phosphorylation is tyrosine 315 (refs 8-10 and unpublished results). The amino acid sequence preceding Tyr 315 includes a tract of six contiguous glutamic acid residues and bears some homology with that preceding the tyrosine phosphorylated in vivo in pp60v-src, the transforming protein of Rous sarcoma virus, and with a region in the polypeptide hormone, gastrin, preceding a tyrosine that is sulphated. Furthermore, although surprisingly large tracts of middle-T may be removed without affecting its transforming activity, mutants that lack the sequences corresponding to amino acids 311-318 inclusive are transformation defective. Because the likely site of phosphorylation, the homology with pp60v-src and gastrin and the sequence apparently required for transformation all overlap, it has generally been accepted that this region of middle-T may form part of an essential region, possibly an active site on the protein. Here we have used techniques of site-directed and site-specific mutagenesis to probe the sequence requirements in more detail. Contrary to expectation, the results obtained strongly suggest that Tyr 315 and conservation of the surrounding amino acid sequence are not essential for transformation.     

一类数列通项的求法

给出对于数列 {un}满足关系式∑ki=0αi(∏n-kj=nj≠n-iuj) =0 的通项求法 .     

一类解非线性方程的高阶解法

本文建立了一类新的解非线性方程一般高阶解法.与牛顿方法和其它方法相比,收敛阶数和效率指数均有所提高.     

A method for haplotype inference in general pedigrees without recombination

The abundance of single nucleotide polymorphisms (SNPs) makes the haplotype-based method instead of single-maker-oriented method the main approach to association studies on QTL mapping. The key problem in haploptype-based method is how to reconstruct haplotypes from genotype data. Directly assaying haplotypes in diploid individuals by experimental methods is too expensive, therefore the in silico haplotyping-determination methods are the major choice at the present. This paper presents a rapid and reliable algorithm for haplotype reconstruction for tightly linked SNPs in general pedigrees. It is based on six rules and consists of three steps. First, the parental origins of alleles in offspring are assigned conditional on genotypes in parent-offspring trios; second, the redundant haplotypes are eliminated based on the six rules; and finally, the most likely haplotype combinations are chosen via maximum likelihood method. Our method was verified and compared with PEDPHASE by simulated data with different pedigree sizes, numbers of loci, and proportions of missing genotypes. The result shows that our algorithm was superior to PEDPHASE in terms of computing time and accuracy of haplotype estimation. The computing time for 100 runs was 10―15 times less and the accuracy was 4%―10% higher than PEDPHASE. The result also indicates that our method was very robust and was hardly affected by pedigree size, number of loci, and proportion of missing genotypes.     

A general method for numerical Green''''s function in arbitrarily layered soils

A straightforward approach is developed to calculate Green's function of a point current source in horizontal multi-layersoils. The sampling value of the coefficient of Green's function is obtained in an iterative way in terms of the equation group satisfying thepertinent boundary value problem. Further, the closed-form expression of multilayered soil Green's function can be given by the vectormatrix pencil technology. The numerical results are in agreement with those by using other softwares. The approach proposed here is ap-plicable to grounding problems with the structure of arbitrarily layered soil without needing the analytical expression of Green's function.     

A unifying model for the G1 period in prokaryotes and eukaryotes

A model to explain the cell division cycle in both prokaryotes and eukaryotes is presented. No specific 'G1 functions' take place during the G1 period, which is merely part of a larger period for the preparation of DNA synthesis which began at the previous initiation of DNA synthesis. A G1 period exists merely because the doubling time of the cells is greater than the sum of the S and G2 periods.     

二阶变系数线性微分方程的不变量解法

用二阶变系数线性微分方程的不变量，给出一种新解法。     

谈求递推数列通项公式的一个方法

《高等数学研究》2006年第一期发表的求递推数列通项公式的一个方法一文给出了关于递推数列通项公式的两个命题很受启发,但命题2有不完整之处,以矩阵为工具对命题2给予完整和推广。     

Accurate prediction of the stability and activity effects of site-directed mutagenesis on a protein core.

Theoretical prediction of the structure, stability and activity of proteins, an important unsolved problem in molecular biology, would be of use for guiding site-directed mutagenesis and other protein-engineering techniques. X-ray diffraction studies have provided extensive structural information for many proteins, challenging theorists to develop reliable techniques able to use such knowledge as a base for prediction of mutants' characteristics. Here we report theoretical calculation of stabilization energies for 78 triple-site sequence variants of lambda repressor characterized experimentally by Lim and Sauer. The calculated energies correlate with the mutants' measured activities; active and inactive mutations are discriminated with 92% reliability. They correlate even more directly with the mutants' thermostabilities, correctly identifying two of the mutants to be more stable than the wild type.     

P137G点突变对嗜热细菌木糖异构酶酶活性及热稳定性的影响

利用重叠延伸法对嗜热细菌（Thermus thermphilus）的木糖异构酶基因xylA进行体外P137G定点突变，通过酿酒酵母表达载体XM204将突变基因xylA137引入酿酒酵母H158中，得到重组菌株H158 XI137. 酶活分析表明，H158 XI137在中温条件下的酶活有很大的提高，与对照菌株H158 XI相比，30?℃时的酶活提高1倍，并且拓宽了最适反应pH，但是其热稳定性大大降低. 结果表明，嗜热细菌木糖异构酶137位的Pro对维持其热稳定性起到重要的作用，并且对其酶学性质有很大的影响.     

生成图的全部极大独立集的一般方法

图的极大独立集问题是图论中重要的NPC问题,独立集具有广泛的应用领域,如编码理论、信道分配、资源配置、纠错码理论等.文章运用拟序关系理论,系统研究了生成图的全部极大独立集的一般方法,该方法简单实用,程序化实现容易.     

协同工作模式下飞机总体布置设计过程建模

针对协同工作模式下传统飞机设计任务分解和过程建模方法存在任务关联语义不明确和复杂流程分析能力弱的不足,提出了一种基于任务关联WBS （Work Breakdown Structure） /Petri网的飞机总体布置设计过程建模方法。在飞机系统WBS基础上,采用可选、必选、选择、因果顺序、协调反馈等五种关系建立任务关联WBS模型,再映射成基于Petri网描述的飞机总体布置设计过程模型,实现飞机总体布置设计任务的静态结构表达和动态流程分析,使得任务分解和过程建模融为一体。通过建立飞机总体布置与协调系统对某飞机总体布置实例进行验证,证明了方法的正确性、有效性和适用性。     

一般约束最优化强收敛的广义强次可行方向法

讨论一般约束最优化,利用广义投影技术和强次可行方向法思想,建立一个初始点任意的新算法,该算法不仅具有全局收敛性,而且是强收敛的,文中还对算法进行数值试验。