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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.
The sectionalized DNA shuffling: an effective tool for molecular directed evolution of Meiothermus ruber TreS
分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

Liu Yan-Chao,Wang Yu-Fan,Qian Ke-Fan,Zhang Jun,Xiao Chen-Peng,Xing Lai-Jun,Li Ming-Chun,

微生物学通报 , 2013,
Abstract: Objective] The gene M-treS from Meiothermus ruber CBS-01 encodes a trehalose synthase of 962 amino acids, named M-TreS. To improve its catalytic activity, we constructed a method of molecular directed evolution, the sectionalized DNA shuffling. Methods] Through two PCR steps with two pairs of partially complementary primers, the M-treS gene was parted into two sections. After the two sections shuffled respectively, a whole gene was obtained through the complementarity of the primers. This method was more feasible, with higher mutability than normal DNA shuffling. Results] Mutants were obtained after one round of the sectionalized DNA shuffling, in combination with error-prone PCR. The best mutant enzyme contained 6 amino acid substitutions, whose catalytic activity and efficiency were 1.6-fold and 2-fold of that of the wild type, respectively. In the 6 amino acid substitutions, 5 were caused by homologous recombination, and one by error-prone PCR. Conclusion] This study indicates that the sectionalized DNA shuffling is an effective tool for molecular directed evolution of macromolecular proteins.
Directed Evolution of D-lactonohydrolase by Error Prone PCR and DNA Shuffling

LIU Zhi-Qiang,SUN Zhi-Hao,ZHENG Pu,LENG Yong,QIAN Jia-Nan,

生物工程学报 , 2005,
Abstract: D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential error prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however, compared with 40% to 20% for wild type.
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,

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.
Evolution of the histones: free play with exon shuffling
Revista chilena de historia natural , 2001, DOI: 10.4067/S0716-078X2001000000020
Abstract: in higher eukaryotes, the nuclear dna is organized for transcription, replication and mitosis in competent chromatin and chromosomes. the basic unit of chromatin is the nucleosome. this entity is formed by 168 base pairs of dna wound around an octamer of histones, this octamer of histones consist of two copies of h2a, h2b, h3 and h4. the dna is sealed in its input and output point by a histone linker: histone h1. histones were supposed to be very conserved proteins. however, during the past few years it was found that these proteins present a high degree of divergency in several lower eukaryotes. in trypanosoma, it was found that histones h3 and h4, which are at the center of the nucleosomal organization, showed more than 30 % of divergency, while histone h1 corresponded to only one of the three peptide domains present in higher eukaryotes. these features of trypanosoma histones may explain, at least in part, the unability of chromatin to condense into chromosomes during the cell division in these parasites. evolution of histones was usually considered as peculiar, with several proposals which are difficult to reconcile with experimental data. in the present work, it is proposed that histones followed the same evolutionary route as many other proteins. considering that exons code for structural and functional domains in proteins and that, at the origin of eukaryotes, the histones, as other proteins, could be formed by "units" (mecano theory), it was expected that these units or domains eventually would be found in living organisms exhibiting primitive features. furthermore, those units could work independently. our results on the structure of trypanosoma cruzi histone genes and proteins as well as the analysis of other histones from different species fit with this proposal
Modeling protein network evolution under genome duplication and domain shuffling
Kirill Evlampiev, Hervé Isambert
BMC Systems Biology , 2007, DOI: 10.1186/1752-0509-1-49
Abstract: We propose and solve a mathematical model of PPI network evolution under successive genome duplications. This demonstrates, from first principles, that evolutionary conservation and scale-free topology are intrinsically linked properties of PPI networks and emerge from i) prevailing exponential network dynamics under duplication and ii) asymmetric divergence of gene duplicates. While required, we argue that this asymmetric divergence arises, in fact, spontaneously at the level of protein-binding sites. This supports a refined model of PPI network evolution in terms of protein domains under exponential and asymmetric duplication/divergence dynamics, with multidomain proteins underlying the combinatorial formation of protein complexes. Genome duplication then provides a powerful source of PPI network innovation by promoting local rearrangements of multidomain proteins on a genome wide scale. Yet, we show that the overall conservation and topology of PPI networks are robust to extensive domain shuffling of multidomain proteins as well as to finer details of protein interaction and evolution. Finally, large scale features of direct and indirect PPI networks of S. cerevisiae are well reproduced numerically with only two adjusted parameters of clear biological significance (i.e. network effective growth rate and average number of protein-binding domains per protein).This study demonstrates the statistical consequences of genome duplication and domain shuffling on the conservation and topology of PPI networks over a broad evolutionary scale across eukaryote kingdoms. In particular, scale-free topologies of PPI networks, which are found to be robust to extensive shuffling of protein domains, appear to be a simple consequence of the conservation of protein-binding domains under asymmetric duplication/divergence dynamics in the course of evolution.Gene duplication is considered the main evolutionary source of new protein functions [1]. Although long suspected [2,3], whole gen
Evolution of Protein Interaction Networks by Whole Genome Duplication and Domain Shuffling  [PDF]
K. Evlampiev,H. Isambert
Quantitative Biology , 2006,
Abstract: Successive whole genome duplications have recently been firmly established in all major eukaryote kingdoms. It is not clear, however, how such dramatic evolutionary process has contributed to shape the large scale topology of protein-protein interaction (PPI) networks. We propose and analytically solve a generic model of PPI network evolution under successive whole genome duplications. This demonstrates that the observed scale-free degree distributions and conserved multi-protein complexes may have concomitantly arised from i) intrinsic exponential dynamics of PPI network evolution and ii) asymmetric divergence of gene duplicates. This requirement of asymmetric divergence is in fact "spontaneously" fulfilled at the level of protein-binding domains. In addition, domain shuffling of multi-domain proteins is shown to provide a powerful combinatorial source of PPI network innovation, while preserving essential structures of the underlying single-domain interaction network. Finally, large scale features of PPI networks reflecting the "combinatorial logic" behind direct and indirect protein interactions are well reproduced numerically with only two adjusted parameters of clear biological significance.
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.
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