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Cloning, expression and functional analysis of the genes in TPS/TPP trehalose synthetic pathway of Meiothermus ruber
红色亚栖热菌TPS/TPP海藻糖合成途径中相关基因的克隆、表达及功能鉴定

Yueming Zhu,Yichen Tang,Hengyi Xu,Juan Zhang,Dongsheng Wei,Laijun Xing,Mingchun Li,
朱玥明
,唐亦辰,徐恒毅,张娟,魏东盛,邢来君,李明春

生物工程学报 , 2009,
Abstract: By constructing the genomic DNA library of Meiothermus ruber CBS-01, the genes of trehalose phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP) involved in trehalose synthesis were cloned. The genes were cloned into the plasmid pET21a, and expressed in Escherichia coli Rosetta gami (DE3). The activities of these two purified enzymes were confirmed by thin layer chromatography (TLC). Meanwhile, we tested the cellular compatible solutes of M. ruber CBS-01 under different environmental pressure,...
Anticipatory evolution and DNA shuffling
Jamie M Bacher, Brian D Reiss, Andrew D Ellington
Genome Biology , 2002, DOI: 10.1186/gb-2002-3-8-reviews1021
Abstract: Proteins are machines created by evolution, but it is unclear just how finely evolution has guided their sequence, structure, and function. It is undoubtedly true that individual mutations in a protein affect both its structure and its function and that such mutations can be fixed during evolutionary history, but it is also true that there are other elements of protein sequence that have been acted upon by evolution. For example, the genetic code appears to be laid out so that mutations and errors in translation are minimally damaging to protein structure and function [1]. Could the probability that a beneficial mutation is found and fixed in the population also have been manipulated during the course of evolution, so that the proteins we see today are more capable of change than the proteins that may have been cobbled together following the 'invention' of translation? Have proteins, in fact, evolved to evolve? There is already some evidence that bacteria are equipped to evolve phenotypes that are more capable of further adaptation (reviewed in [2,3,4]). For example, mutator [5] and hyper-recombinogenic [6] strains arise as a result of selection experiments. The development of DNA shuffling (reviewed in [7,8]) and the appearance of several recent papers using this technique [9,10,11] provide us with a surprising new opportunity to ask and answer these fundamental questions at the level of individual genes, and perhaps even genomes.DNA shuffling, a method for in vitro recombination, was developed as a technique to generate mutant genes that would encode proteins with improved or unique functionality [12,13]. It consists of a three-step process that begins with the enzymatic digestion of genes, yielding smaller fragments of DNA. The small fragments are then allowed to randomly hybridize and are filled in to create longer fragments. Ultimately, any full-length, recombined genes that are recreated are amplified via the polymerase chain reaction. If a series of alleles o
A novel method of DNA shuffling without PCR process
Qiang Wang,Qiuyun Liu,Gang Li,Baojian Li
Chinese Science Bulletin , 2004, DOI: 10.1007/BF03184266
Abstract: Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends. These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.
A novel method of DNA shuffling without PCR process
WANG Qiang,LIU Qiuyun,LI Gang & LI Baojian Key Laboratory of Gene Engineering of Ministry of Education,School of Life Sciences,Zhongshan University,Guangzhou,China Correspondence should be addressed to Li Baojian,
WANGQiang
,LIUQiuyun,LIGang,LIBaojian

科学通报(英文版) , 2004,
Abstract: Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends.These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.
Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes  [PDF]
Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005553
Abstract: We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
Analytical study of the effect of recombination on evolution via DNA shuffling  [PDF]
Weiqun Peng,Herbert Levine,Terence Hwa,David A. Kessler
Physics , 2003, DOI: 10.1103/PhysRevE.69.051911
Abstract: We investigate a multi-locus evolutionary model which is based on the DNA shuffling protocol widely applied in \textit{in vitro} directed evolution. This model incorporates selection, recombination and point mutations. The simplicity of the model allows us to obtain a full analytical treatment of both its dynamical and equilibrium properties, for the case of an infinite population. We also briefly discuss finite population size corrections.
Directed DNA Shuffling of Retrovirus and Retrotransposon Integrase Protein Domains  [PDF]
Xiaojie Qi, Edwin Vargas, Liza Larsen, Whitney Knapp, G. Wesley Hatfield, Richard Lathrop, Suzanne Sandmeyer
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0063957
Abstract: Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.
一种方便、无PCR过程的DNA shuffling方法
王强,刘秋云,李刚,李宝健
科学通报 , 2004,
Abstract: 目前大多数的DNA shuffling方法都需要PCR过程.由于一些限制内切酶的识别位点与切割位点不重叠而产生不同的黏性末端,当再连接的时候,同源片段之间就可以混合起来按照正确顺序组装成新的嵌合体分子,利用这种性质,构建了一种新的、无PCR过程的、简便的shuffling方法.
Preparation of PFS coagulant by sectionalized reactor
CHANG Qing,WANG Hong-yu,
CHANG Qing
,WANG Hong-yu

环境科学学报(英文版) , 2002,
Abstract: The oxidation rate of ferrous sulfate is investigated for the preparation of polyferric sulfate(PFS) coagulant. It is proved that this reaction is zero order with respect to Fe 2+ , first order with respect to NO 2(g), and first order with respect to the interface area between gas phase and liquid phase. According to this mechanism, sectionalized reactor(SR) is used in place of traditional reactor(TR), and the liquid of reaction mixture is recycled by pump. As a result, not only the flow path of reaction liquid is prolonged, but also gas liquid contact area enlarged, and the reaction distinctly accelerated, compared with traditional reactor. The effects of parameters including temperature, acidity and others on the reaction rate are also discussed.
Extensive domain shuffling in transcription regulators of DNA viruses and implications for the origin of fungal APSES transcription factors
Lakshminarayan M Iyer, Eugene V Koonin, L Aravind
Genome Biology , 2002, DOI: 10.1186/gb-2002-3-3-research0012
Abstract: The phyletic range and the conserved DNA-binding domains of the viral regulatory proteins of the poxvirus D6R/N1R and baculoviral Bro protein families have not been previously defined. Using computational analysis, we show that the amino-terminal module of the D6R/N1R proteins defines a novel, conserved DNA-binding domain (the KilA-N domain) that is found in a wide range of proteins of large bacterial and eukaryotic DNA viruses. The KilA-N domain is suggested to be homologous to the fungal DNA-binding APSES domain. We provide evidence for the KilA-N and APSES domains sharing a common fold with the nucleic acid-binding modules of the LAGLIDADG nucleases and the amino-terminal domains of the tRNA endonuclease. The amino-terminal module of the Bro proteins is another, distinct DNA-binding domain (the Bro-N domain) that is present in proteins whose domain architectures parallel those of the KilA-N domain-containing proteins. A detailed analysis of the KilA-N and Bro-N domains and the associated domains points to extensive domain shuffling and lineage-specific gene family expansion within DNA virus genomes.We define a large class of novel viral DNA-binding proteins and their cellular homologs and identify their domain architectures. On the basis of phyletic pattern analysis we present evidence for a probable viral origin of the fungus-specific cell-cycle regulatory transcription factors containing the APSES DNA-binding domain. We also demonstrate the extensive role of lineage-specific gene expansion and domain shuffling, within a limited set of approximately 24 domains, in the generation of the diversity of virus-specific regulatory proteins.Large DNA viruses of bacteria and eukaryotes have complex life cycles with several distinct phases that involve diverse virus-host interactions. An array of regulatory systems mediate activation or repression of expression of specific batteries of viral genes that are required at different phases of the life cycle. Other sets of regu
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