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1.
《Reviews in Molecular Biotechnology》2000,74(4):303-315
The creation of diversity in populations of polypeptides has become an important tool in the derivation of polypeptides with useful characteristics. This requires efficient methods to create diversity coupled with methods to select polypeptides with desired properties. In this review we describe the use of in vivo recombination as a powerful way to generate diversity. The novel principles for the recombination process and several applications of this process for the creation of phage antibody libraries are described. The advantage and disadvantages are discussed and possible future exploitation presented. 相似文献
2.
Enzyme optimization: moving from blind evolution to statistical exploration of sequence-function space 总被引:2,自引:0,他引:2
Directed evolution is a powerful tool for the creation of commercially useful enzymes, particularly those approaches that are based on in vitro recombination methods, such as DNA shuffling. Although these types of search algorithms are extraordinarily efficient compared with purely random methods, they do not explicitly represent or interrogate the genotype-phenotype relationship and are essentially blind in nature. Recently, however, researchers have begun to apply multivariate statistical techniques to model protein sequence-function relationships and guide the evolutionary process by rapidly identifying beneficial diversity for recombination. In conjunction with state-of-the-art library generation methods, the statistical approach to sequence optimization is now being used routinely to create enzymes efficiently for industrial applications. 相似文献
3.
Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating “smart” libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field. 相似文献
4.
Meiotic recombination is almost universal among sexually reproducing organisms. Because the process leads to the destruction of successful parental allele combinations and the creation of novel, untested genotypes for offspring, the evolutionary forces responsible for the origin and maintenance of this counter-intuitive process are still enigmatic. Here, we have used newly available genetic data to compare genome-wide recombination rates in a report on recombination rates among different taxa. In particular, we find that among the higher eukaryotes exceptionally high rates are found in social Hymenoptera. The high rates are compatible with current hypotheses suggesting that sociality in insects strongly selects for increased genotypic diversity in worker offspring to either meet the demands of a sophisticated caste system or to mitigate against the effects of parasitism. Our findings might stimulate more detailed research for the comparative study of recombination frequencies in taxa with different life histories or ecological settings and so help to understand the causes for the evolution and maintenance of this puzzling process. 相似文献
5.
Exploiting recombination in single bacteria to make large phage antibody libraries 总被引:33,自引:0,他引:33
The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens. 相似文献
6.
Damien Biot-Pelletier Vincent J. J. Martin 《Applied microbiology and biotechnology》2014,98(9):3877-3887
An upsurge in the bioeconomy drives the need for engineering microorganisms with increasingly complex phenotypes. Gains in productivity of industrial microbes depend on the development of improved strains. Classical strain improvement programmes for the generation, screening and isolation of such mutant strains have existed for several decades. An alternative to traditional strain improvement methods, genome shuffling, allows the directed evolution of whole organisms via recursive recombination at the genome level. This review deals chiefly with the technical aspects of genome shuffling. It first presents the diversity of organisms and phenotypes typically evolved using this technology and then reviews available sources of genetic diversity and recombination methodologies. Analysis of the literature reveals that genome shuffling has so far been restricted to microorganisms, both prokaryotes and eukaryotes, with an overepresentation of antibiotics- and biofuel-producing microbes. Mutagenesis is the main source of genetic diversity, with few studies adopting alternative strategies. Recombination is usually done by protoplast fusion or sexual recombination, again with few exceptions. For both diversity and recombination, prospective methods that have not yet been used are also presented. Finally, the potential of genome shuffling for gaining insight into the genetic basis of complex phenotypes is also discussed. 相似文献
7.
Meiotic recombination has two key functions: the faithful assortment of chromosomes into gametes and the creation of genetic diversity. Both processes require that meiotic recombination occurs between homologous chromosomes, rather than sister chromatids. Accordingly, a host of regulatory factors are activated during meiosis to distinguish sisters from homologs, suppress recombination between sister chromatids and promote the chromatids of the homologous chromosome as the preferred recombination partners. Here, we discuss the recent advances in our understanding of the mechanistic basis of meiotic recombination template choice, focusing primarily on developments in the budding yeast, Saccharomyces cerevisiae, where the regulation is currently best understood. 相似文献
8.
Over the past 20 years, directed evolution has been seen to be the most reliable approach to protein engineering. Emulating the natural selection algorithm, ad hoc enzymes with novel features can be tailor-made for practical purposes through iterative rounds of random mutagenesis, DNA recombination and screening. Of the heterologous hosts used in laboratory evolution experiments, the budding yeast Saccharomyces cerevisiae has become the best choice to express eukaryotic proteins with improved properties. S. cerevisiae not only allows mutant enzymes to be secreted but also, it permits a wide range of genetic manipulations to be employed, ranging from in vivo cloning to the creation of greater molecular diversity, thanks to its efficient DNA recombination apparatus. Here, we summarize some successful examples of the use of the S. cerevisiae machinery to accelerate artificial evolution, complementing the traditional in vitro methods to generate tailor-made enzymes. 相似文献
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Conventional phylogenetic tree estimation methods assume that all sites in a DNA multiple alignment have the same evolutionary history. This assumption is violated in data sets from certain bacteria and viruses due to recombination, a process that leads to the creation of mosaic sequences from different strains and, if undetected, causes systematic errors in phylogenetic tree estimation. In the current work, a hidden Markov model (HMM) is employed to detect recombination events in multiple alignments of DNA sequences. The emission probabilities in a given state are determined by the branching order (topology) and the branch lengths of the respective phylogenetic tree, while the transition probabilities depend on the global recombination probability. The present study improves on an earlier heuristic parameter optimization scheme and shows how the branch lengths and the recombination probability can be optimized in a maximum likelihood sense by applying the expectation maximization (EM) algorithm. The novel algorithm is tested on a synthetic benchmark problem and is found to clearly outperform the earlier heuristic approach. The paper concludes with an application of this scheme to a DNA sequence alignment of the argF gene from four Neisseria strains, where a likely recombination event is clearly detected. 相似文献
11.
Vergin KL Tripp HJ Wilhelm LJ Denver DR Rappé MS Giovannoni SJ 《Environmental microbiology》2007,9(10):2430-2440
Recombination is an important process in microbial evolution. Rates of recombination with extracellular DNA matter because models of microbial population structure are profoundly influenced by the degree to which recombination is occurring within the population. Low rates of recombination may be sufficient to ensure the lateral propagation of genes that have a high selective advantage without disrupting the clonal pattern of inheritance for other genes. High rates of recombination potentially can obscure clonal patterns, leading to linkage equilibrium, and give microbial populations a population genetic structure more akin to sexually interbreeding eukaryotic populations. We examined eight loci from nine strains of candidatus Pelagibacter ubique (SAR11), isolated from a single 2L niskin sample of natural seawater, for evidence of genetic recombination between strains. The Shimodaira-Hasegawa test revealed significant phylogenetic incongruence in seven of the genes, indicating that frequent recombination obscures phylogenetic signals from the linear inheritance of genes in this population. Statistical evidence for intragenic recombination was found for six loci. An informative sites matrix showed extensive evidence for a widespread breakdown of linkage disequilibrium. Although the mechanisms of genetic transfer in native SAR11 populations are unknown, we measured recombination rates, rho, that are much higher than point mutation rates, theta, as a source of genetic diversity in this clade. The eukaryotic model of species sharing a common pool of alleles is more apt for this SAR11 population than a strictly clonal model of inheritance in which allelic diversity is controlled by periodic selection. 相似文献
12.
Indira Umareddy Olivier Pluquet Qing Yin Wang Subhash G Vasudevan Eric Chevet Feng Gu 《Virology journal》2007,4(1):1-10
Background
Tomato-infecting begomoviruses are widely distributed across the world and cause diseases of high economic impact on wide range of agriculturally important crops. Though recombination plays a pivotal role in diversification and evolution of these viruses, it is currently unknown whether there are differences in the number and quality of recombination events amongst different tomato-infecting begomovirus species. To examine this we sought to characterize the recombination events, estimate the frequency of recombination, and map recombination hotspots in tomato-infecting begomoviruses of South and Southeast Asia.Results
Different methods used for recombination breakpoint analysis provided strong evidence for presence of recombination events in majority of the sequences analyzed. However, there was a clear evidence for absence or low Recombination events in viruses reported from North India. In addition, we provide evidence for non-random distribution of recombination events with the highest frequency of recombination being mapped in the portion of the N-terminal portion of Rep.Conclusion
The variable recombination observed in these viruses signified that all begomoviruses are not equally prone to recombination. Distribution of recombination hotspots was found to be reliant on the relatedness of the genomic region involved in the exchange. Overall the frequency of phylogenetic violations and number of recombination events decreased with increasing parental sequence diversity. These findings provide valuable new information for understanding the diversity and evolution of tomato-infecting begomoviruses in Asia. 相似文献13.
A neutral explanation for the correlation of diversity with recombination rates in humans 总被引:38,自引:0,他引:38
Hellmann I Ebersberger I Ptak SE Pääbo S Przeworski M 《American journal of human genetics》2003,72(6):1527-1535
One of the most striking findings to emerge from the study of genomic patterns of variation is that regions with lower recombination rates tend to have lower levels of intraspecific diversity but not of interspecies divergence. This uncoupling of variation within and between species has been widely interpreted as evidence that natural selection shapes patterns of genetic variability genomewide. We revisited the relationship between diversity, divergence, and recombination in humans, using data from closely related species and better estimates of recombination rates than previously available. We show that regions that experience less recombination have reduced divergence to chimpanzee and to baboon, as well as lower levels of diversity. This observation suggests that mutation and recombination are associated processes in humans, so that the positive correlation between diversity and recombination may have a purely neutral explanation. Consistent with this hypothesis, diversity levels no longer increase significantly with recombination rates after correction for divergence to chimpanzee. 相似文献
14.
Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination 总被引:59,自引:0,他引:59
A purified preparation of the Escherichia coli integration host factor (IHF) displays two polypeptides of apparent molecular weight 11,000 and 9,500 when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Under nondenaturing conditions, IHF appears to exist as a 1:1 complex of these two polypeptides. Integrative recombination takes place in vitro when purified IHF and purified Int, a product of a bacteriophage lambda gene, are the only proteins added to reaction mixtures. No recombination is detected in the absence of either protein. The characteristics of the recombination reaction carried out by these two purified proteins are described. Purified IHF binds to DNA; in the presence of Int, a ternary complex is formed at one of the specific recombination sites. IHF hs no detectable endonuclease or topoisomerase activity. Several possibilities for the role of IHF in recombination are considered. 相似文献
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16.
Directed evolution experiments rely on the cyclical application of mutagenesis, screening and amplification in a test tube. They have led to the creation of novel proteins for a wide range of applications. However, directed evolution currently requires an uncertain, typically large, number of labor intensive and expensive experimental cycles before proteins with improved function are identified. This paper introduces predictive models for quantifying the outcome of the experiments aiding in the setup of directed evolution for maximizing the chances of obtaining DNA sequences encoding enzymes with improved activities. Two methods of DNA manipulation are analysed: error-prone PCR and DNA recombination. Error-prone PCR is a DNA replication process that intentionally introduces copying errors by imposing mutagenic reaction conditions. The proposed model calculates the probability of producing a specific nucleotide sequence after a number of PCR cycles. DNA recombination methods rely on the mixing and concatenation of genetic material from a number of parent sequences. This paper focuses on modeling a specific DNA recombination protocol, DNA shuffling. Three aspects of the DNA shuffling procedure are modeled: the fragment size distribution after random fragmentation by DNase I, the assembly of DNA fragments, and the probability of assembling specific sequences or combinations of mutations. Results obtained with the proposed models compare favorably with experimental data. 相似文献
17.
Testing for effects of recombination rate on nucleotide diversity in natural populations of Arabidopsis lyrata 总被引:3,自引:0,他引:3
Wright SI Foxe JP DeRose-Wilson L Kawabe A Looseley M Gaut BS Charlesworth D 《Genetics》2006,174(3):1421-1430
We investigated DNA sequence diversity for loci on chromosomes 1 and 2 in six natural populations of Arabidopsis lyrata and tested for the role of natural selection in structuring genomewide patterns of variability, specifically examining the effects of recombination rate on levels of silent polymorphism. In contrast with theoretical predictions from models of genetic hitchhiking, maximum-likelihood-based analyses of diversity and divergence do not suggest reduction of diversity in the region of suppressed recombination near the centromere of chromosome 1, except in a single population from Russia, in which the pericentromeric region may have undergone a local selective sweep or demographic process that reduced variability. We discuss various possibilities that might explain why nucleotide diversity in most A. lyrata populations is not related to recombination rate, including genic recombination hotspots, and low gene density in the low recombination rate region. 相似文献
18.
All major regions of FtsK are required for resolution of chromosome dimers 总被引:3,自引:0,他引:3
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Resolution of chromosome dimers, by site-specific recombination between dif sites, is carried out in Escherichia coli by XerCD recombinase in association with the FtsK protein. We show here that a variety of altered FtsK polypeptides, consisting of the N-terminal (cell division) domain alone or with deletions in the proline-glutamine-rich part of the protein, or polypeptides consisting of the C-terminal domain alone are all unable to carry out dif recombination. Alteration of the putative nucleotide-binding site also abolishes the ability of FtsK to carry out recombination between dif sites. 相似文献
19.
Many eukaryotic proteins exist in large multisubunit assemblies and often show compromised folding or activity when their
interaction partners are not present. Protein complexes in eukaryotes can contain ten or more subunits with individual polypeptides
ranging in size up to several hundred kilodalton, severely restricting the application of conventional cloning strategies
and imposing constraints on the choice of the expression host. Modern structural molecular biology often depends on introducing
diversity into the specimens under investigation, including mutation, truncation and placement of purification aids. Current
recombinant expression methods often require a disproportionate labor investment prior to multiprotein expression, and subsequent
to expression and analysis do not provide for rapid revision of the experiment. We have developed reagents and protocols for
rapid and flexible multiprotein complex expressions suitable for structural biology, focusing on multigene baculoviral vectors
and their recombination mediated assembly. A top priority in protein science is automation. Our strategy can be readily adapted
in a robotics setup, for baculovirus/insect cell expression of protein complexes, but likewise also for mammalian or prokaryotic
hosts. 相似文献
20.
Many plants combine sexual reproduction with some form of asexual reproduction to different degrees, and lower genetic diversity is expected with asexuality. Moreover, the ratios of sexual morphs in species with gender dimorphism are expected to vary in proportion to the reproductive success of the sexual process. Hence, sex ratios can directly influence the genetic structure and diversity of a population. We investigated genotypic diversity in 23 populations of a facultative, apomictic gynodioecious orchid, Satyrium ciliatum, to examine the effect on genotypic diversity of variation in the frequency of females and in the amount of sexual reproduction. The study involved one pure female, seven gynodioecious (both females and hermaphrodites present) and 15 hermaphroditic populations. Pollinia receipt was higher in hermaphroditic than in gynodioecious populations. Analyses of variation in ISSRs demonstrated that genotypic diversity was high in all populations and was not significantly different between hermaphroditic and gynodioecious populations. We used character compatibility analysis to determine the extent to which recombination by sexual reproduction contributed to genotypic diversity. The results indicate that the contribution of recombination to genotypic diversity is higher in hermaphroditic than in gynodioecious populations, consistent with the finding that hermaphroditic populations received higher amounts of pollinia. Our finding of reduced recombination in gynodioecious populations suggests that maintenance of sex in hermaphrodites plays an important role in generating genotypic diversity in this apomictic orchid. 相似文献