A second-generation system for unbiased reading frame selection

Reading frame selection of nucleic acids has important implications for protein engineering and genomics. Current methods are limited because selection of the gene of interest inevitably depends on the solubility of its translated product. Here we report the construction of the pInSALect vector, which provides strict reading frame selection without concomitant selection for protein solubility or folding. This plasmid incorporates the cis-splicing VMA intein sequence from Saccharomyces cerevisiae to facilitate the post-translational self-excision of the protein of interest, thereby eliminating potential aggregation problems. Results from two libraries of chimeric glycinamide ribonucleotide formyltransferases confirm the superior performance of pInSALect over existing reading frame selection systems.     

水溶液晶体的空间生长及其地基模拟实验

空间水溶液晶体生长是通过搭载人造卫星、宇宙飞船和空间站等飞行器,在微重力条件下从水溶液中进行晶体生长实验.许多透明单晶体,诸如:KDP(磷酸二氢钾,potassium dihydrogen phosphate)、ADP(磷酸二氢铵,ammonium dihydrogen phosphate)、TGS(硫酸三甘氨酸,triglycine sulfae)、沸石和α-碘酸锂(α-LiIO3)等无机非线性光学晶体,尿素等有机非线性光学晶体以及蛋白质等生物晶体,均可采用水溶液法生长.美国、前苏联、西欧和日本等国在从事空间材料科学研究的高科技发展计划中,均把水溶液晶体生长作为一项重要项目.由于水溶液晶体生长方法的研究在地面上已有丰富的基础,而且具有温度低,能耗小,可实现原位实时观察等优点,所以,它是探索空间晶体生长原理和方法、研究晶体生长微重力效应的重要材料制备技术.由于空间晶体生长受搭载条件限制,空间实验的次数与地面相比是十分稀少的,为了获得空间实验的高成功率,在地面上必须建立相应的实验基地,开展深入的地基研究工作.本文将分别从空间实验和地基研究两个方面作系列介绍,包括蛋白质晶体,沸石晶体,α-LiIO3晶体的空间生长以及TGS,NaClO3(sodium chlorate,氯酸钠),Sr(NO3)2(strontium nitrate,硝酸鍶)和Ba(NO3)2(barium nitrate,硝酸钡)等晶体生长过程的原位实时观察,并展望了空间晶体生长的发展前景.     

Analysis of the Insect OS-D-Like Gene Family

Insect OS-D-like proteins, also known as chemosensory (CSP) or sensory appendage proteins (SAP), are broadly expressed in various insect tissues, where they are thought to bind short to medium chain length fatty acids and their derivatives. Although their specific function remains uncertain, OS-D-like members have been isolated from sensory organs (including the sensillum lymph in some cases), and a role in olfaction similar to that of the insect odorant binding proteins (OBP) has been suggested for some. We have identified 15 new OS-D-like sequences: four from cDNA clones described herein and 11 from sequence databases. The os-d-like genes from the Anopheles gambiae, Apis mellifera, Drosophila melanogaster, and Drosophila pseudoobscura genomes typically have single, small introns with a conserved splice site. Together with all family members entered on GenBank, a total of 70 OS-D-like proteins, representing the insect orders Diptera, Dictyoptera, Hymenoptera, Lepidoptera, Orthoptera, and Phasmatodea, were analyzed. A neighbor joining distance phenogram identified several protein similarity classes that were characterized by highly conserved sequence motifs, including (A) N-terminal YTTKYDN(V/I)(N/D)(L/V)DEIL, (B) central DGKELKXX(I/L)PDAL, and (C) C-terminal KYDP. In contrast, three similarity classes were characterized by their diversion from these conserved motifs. The functional importance of conserved amino acid residues is discussed in relation to the crystal and NMR structures of MbraCSPA6.     

Synthetic hydrogels: 3. Hydroxyalkyl acrylate and methacrylate copolymers: surface and mechanical properties

The surface and mechanical properties of copolymers of hydroxyalkyl acrylates and methacrylates have been examined by a variety of techniques. This work is complementary to earlier parts of this series which describe the effect of copolymer structure on water binding properties. Water structure has been demonstrated to exert a profound effect upon mechanical properties whether measured in compression or in tension. In particular, water that is characterized by differential scanning calorimetry as ‘freezing’ water is observed to have a marked plasticizing effect upon the gel, whereas ‘non-freezing’ water has little such effect. Similarly, the ‘freezing’ water produces a more marked effect on thermally induced transitions. Two distinct transition points are observed as a result of its presence. One corresponds to the freezing point of water and the other to a glass transition temperature, whose value depends upon the proportion of ‘freezing’ or ‘plasticizing’ water in the gel. Several predictive and direct measurement techniques have been used to study the surface properties of the copolymers in both hydrated and dehydrated states. Taken together they have established a sound understanding of the way in which polar and dispersive components of surface free energy vary as a function of copolymer composition and water content. Use of protein adsorption and fibroblast cell interaction techniques demonstrate that biological phenomena respond to changes at a molecular level which current macroscopic surface energy techniques are unable to discern.     

Thermoplastic extrusion—the mechanism of the formation of extrudate structure and properties

Systems processed by thermoplastic extrusion can be regarded as heterophase polymer melts of incompatible water-plasticized biopolymers. In the process of thermoplastic extrusion, proteins and polysaccharides are melted at high pressure and temperature below the temperature region of their thermal decomposition. Dispersed particles of these systems can be deformed in flow. The mixed-melt anisotropic structure, formed in flow, is fixed by rapid conversion of the melt jet that lets the extruder die from a viscous state to a rubber-like state and then to a glassy state caused by cooling and drying. Incompatibility of proteins and polysaccharides in their water-plasticized melt mixtures impacts on structure formation and texturization during thermoplastic extrusion. Presented at the 20th ISF World Congress and 83rd AOCS Annual Meeting and Expo, May 10–14, 1992, Toronto, Ontario, Canada.     

Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Context dependence of phenotype prediction and diversity in combinatorial mutagenesis

Two different combinatorial mutagenesis experiments on the light-harvestingII (LH2) protein of Rhodobacter capsulatus indicate that heuristicrules relating sequence directly to phenotype are dependenton which sets or groups of residues are mutated simultaneously.Previously reported combinatorial mutagenesis of this chromogenicprotein (based on both phylogenetic and structural models) showedthat substituting amino acids with large molar volumes at Gly^ß31caused the mutated protein to have a spectrum characteristicof light-harvesting I (LH1). The six residues that underwentcombinatorial mutagenesis were modeled to lie on one side ofa transmembrane -helix that binds bacteriochlorophyll. In asecond experiment described here, we have not used structuralmodels or phylogeny in choosing mutagenesis sites. Instead,a set of six contiguous residues was selected for combinatorialmutagenesis. In this latter experiment, the residue substitutedat Gly^ß31 was not a determining factor in whetherLH2 or LH1 spectra were obtained; therefore, we conclude thatthe heuristic rules for phenotype prediction are context dependent.While phenotype prediction is context dependent, the abilityto identify elements of primary structure causing phenotypediversity appears not to be. This strengthens the argument forperforming combinatorial mutagenesis with an arbitrary groupingof residues if structural models are unavailable.     

Effect of birch (Betula pendula) bark and food protein level on root voles (Microtus oeconomus): II. detoxification capacity

The effect of protein and birch bark powder (BBP) content of forage on detoxification capacity of root voles (Microtus oeconomus) was studied. Young voles were fed with eight different diets for two weeks. Individuals on low (3%) protein diets had significantly lighter livers and kidneys than those on moderate (6%) or high (12%) protein diets. Birch bark powder addition did not have significant effect on organ weight. Detoxification was significantly induced, apparently due to secondary compounds in BBP. The activity of ethoxyresorufin-O-dealkylase (EROD) was high when protein content or BBP concentration in forage was high as compared to low protein diets or diets containing no BBP. Glucuronidation, on the other hand, was not induced by BBP. High BBP content caused serious physiological stress to the voles. The only individuals surviving were those capable of sufficiently allocating energy and protein to detoxification.     

Pattern-induced multi-sequence alignment (PUMA) algorithm employing secondary structure-dependent gap penalties for use in comparative protein modelling

A multiple sequence alignment algorithm is described that usesa dynamic programming-based pattern construction method to aligna set of homologous sequences based on their common patternof conserved sequence elements. This pattern-induced multi-sequencealignment (PUMA) algorithm can employ secondary-structure dependentgap penalties for use in comparative modelling of new sequenceswhen the three-dimensional structure of one or more membersof the same family is known. We show that the use of secondarystructure information can significantly improve the accuracyof aligning structure boundaries in a set of homologous sequenceseven when the structure of only one member of the family isknown     

现代生物工程技术简介

对组成现代生物工程的六大技术系统：基因工程、蛋白质工程、酶工程、细胞工程、微生物工程、生化工程的历史和现状，工作内容及应用前景作了简明的介绍。