Expression of the catalytic subunits of pol α and pol δ from fission yeast Schizosaccharomyces pombe

This paper reports on expression and posttranslational modifications of the catalytic subunits of pol α and pol δ from fission yeastSchizosaccharomyces pombe. Okadaic acid treatment ofS. pombe spheroplasts in amounts known to inhibit phosphatases, 1 and 2A resulted in decreased proteolysis of both pol α and pol δ. Computer analysis of pol α and pol δ sequences confirmed the presence of consensus motifs for protein phosphorylation. Indirect immunofluorescence microscopy ofS. pombe cells showed nuclear location of both proteins in wild type cells. However, whereas cells transformed with a vector expressing pol α produced a clear increase of the nuclear signal no increase was detectable in cells transformed with pol δ. This observation suggests the existence of a mechanism limiting thecell concentration of pol δ in the cell. Constitutive expression ofS. pombe pol δ inE. coli was possible only with vectors containing truncated forms of its gene, indicating a toxic effect of pol δ onE. coli growth.     

人类polⅡ启动子的识别

依据基因启动子区和非启动子区碱基分布的特征,应用基于多样性增量的二次判别分析 (IDQD),对人类polⅡ启动子进行识别,识别精度达到90％以上的水平,优于其他已发表的 (包括SVM分类器等) 识别算法. 使用IDQD算法也能对转录起始位点 (TSS) 进行较准确的预测,10-fold交叉检验结果的敏感性和特异性分别为86％和91％. 这些结果表明IDQD是一个有效的分类器.     

理论预测果蝇polⅡ启动子

基于果蝇polⅡ启动予的序列特征,利用结合了离散增量和位置权重矩阵的贝叶斯判别函数对果蝇启动予进行了预测。对预测算法进行10交叉检验。通过比较不同大小训练集对结果的影响,说明了参数选取的合理性和算法的预测能力。同时比较了不同参数的选取对预测结果的影响,从而获得最佳启动子预测结果。预测结果显示成功率达到93％,相互关联系数达到83％。     

The primary DNA-binding subsite of the rat pol β. Energetics of interactions of the 8-kDa domain of the enzyme with the ssDNA

Interactions of the 8-kDa domain of the rat pol β and the intact enzyme with the ssDNA have been studied, using the quantitative fluorescence titration technique. The 8-kDa domain induces large topological changes in the bound DNA structure and engages much larger fragments of the DNA than when embedded in the intact enzyme. The DNA affinity of the domain is predominantly driven by entropy changes, dominated by the water release from the protein. The thermodynamic characteristics dramatically change when the domain is embedded in the intact polymerase, indicating the presence of significant communication between the 8-kDa domain and the catalytic 31-kDa domain. The diminished water release from the 31-kDa domain strongly contributes to its dramatically lower DNA affinity, as compared to the 8-kDa domain. Unlike the 8-kDa domain, the DNA binding of the intact pol β is driven by entropy changes, originating from the structural changes of the formed complexes.     

Structure of the δ-Chain of Chimpanzee Haemoglobin A<Subscript>2</Subscript>

STUDIES of adult¹ and foetal² haemoglobin from the chimpanzee (Pan troglodytes) have shown that the amino-acid compositions of tryptic and chymotryptic peptides of the α, β and γ-chains are indistinguishable from those of man. The primary structures of chimpanzee α, β and γ-chains are therefore almost certainly identical to the homologous human chains. The two types of γ-chains found in man³, ^Gγ and ^Aγ, with glycine and alanine in position γ136, respectively, are likewise present in the chimpanzee².     

Effects of antimicrobial activity of silver nanoparticles on in vitro establishment of G × N15 (hybrid of almond × peach) rootstock

In the present investigation were evaluated the antifungal and antibacterial activities of Nano-silver (NS). Two separate experiments were done to evaluate the potential of silver nanoparticles in controlling the contamination of G × N15 micro-propagation. In the first experiment, a factorial experiment based on a completely randomized design with 15 treatments including five different NS concentrations (0, 50, 100, 150 and 200 ppm) and three soaking time of explants (3, 5 and 7 min) with five replications was conducted. In the other experiment, NS was added to Murashige and Skoog (MS) medium with concentrations of 0, 50, 100, 150 and 200 ppm in a completely randomized design. Results showed that the application of 100 and 150 ppm NS both as an immersion and as added directly to the culture medium significantly reduces internal and external contaminations compared with the control group. Using NS in culture medium was more efficient to reduce fungal and bacterial contamination than immersion. High concentrations of NS had an adverse effect on the viability of G × N15 single nodes and this effect was more serious in immersed explants in NS than directly added NS ones regarding the viability of buds and plantlet regeneration. In conclusion, due to high contamination of woody plants especially fruit trees and also adverse environmental effects of mercury chloride, the NS solution can be used as a low risk bactericide in micro-propagation of G × N15 and can be an appropriate alternative to mercury chloride in the future.     

Can the combination of electrochemical regeneration of NAD+, selectivity of L‐α‐amino‐acid dehydrogenase,and reductive amination of α‐keto‐acid be applied to the inversion of configuration of a L‐α‐amino‐acid?

The inversion of configuration of L‐alanine can be carried out by combining its selective oxidation in the presence of NAD⁺ and L‐alanine dehydrogenase, electrochemical regeneration of the NAD⁺ at a carbon felt anode, and reductive amination of pyruvate, i.e., reduction of its imino derivative at a mercury cathode, the reaction mixture being buffered with concentrated ammonium/ammonia (1.28M / 1.28M). The dehydrogenase exhibits astonishing activity and stability under such extreme conditions of pH and ionic strength. The main drawback of the process is its slowness. At best, the complete inversion of a 10 mM solution of L‐alanine requires 140 h. A careful and detailed quantitative analysis of each of the key steps involved shows that the enzyme catalyzed oxidation is so thermodynamically uphill that it can be driven efficiently to completion only when both the coenzyme regeneration and the pyruvate reduction are very effective. The first condition is easily fulfilled. Under the best conditions, it is the rate of the chemical reaction producing the imine which controls the whole process kinetically. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 101–107, 1999.     

Study of the Mechanism of Interaction of Oligonucleotides with the 3′-Terminal Region of tRNA<Superscript>Phe</Superscript> by Computer Modeling

Three-dimensional atomic models of complexes between yeast tRNA^Phe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNA^Phe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNA^Phe destruction and formation of the final tRNA^Phe-oligonucleotide complementary complex. The primary complex formation and the following tRNA^Phe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element.     

Computational studies of the binding site of α<Subscript>1A</Subscript>-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human α_1A-adrenoceptor (α_1A-AR), we have successfully developed its homology model based on the crystal structure of β₂-AR. Subsequent structural refinements were performed to mimic the receptor’s natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective α_1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective α_1A-AR antagonists and for guiding further mutagenesis studies. Figure The superposition of β₂-AR crystal structure (gold ribbons) and α_1A-AR homology model (blue ribbons)     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

An Empirical Potential Study of the Interaction of L—;Lysine—;L—;Alanine—;L—;Alanine Tripeptide with Four models of B—;DNA with different Compositions

Abstract

The empirical potential including the intra- and intermolecular energy terms was used to study the interaction of L-Lysine-L-Alanine-L-Alanine Tripeptide with four models of B-DNA with different compositions. On the basis of a detailed search of the respective potential energy surface, it was found that the peptide is preferentially bounded to the AT-rich sequences. Analysis of the different energy contributions indicated that the electrostatic term is responsible for this preference. The results agree with the experimental data on the selectivity of some DNA—binding proteins and polypeptides to AT—;rich DNA.