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At least 1,009 records · Page 56Linked to original sources
PubMed2005

Reducing mutational bias in random protein libraries.

The success of protein optimization through directed molecular evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by separate error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate numbers of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concentration of template in the shuffling reaction while taking into account the GC content of the target gene.

Bacillus thuringiensis↗
PubMed2005

Engineering of Kex2 variants exhibiting altered substrate specificity.

Engineering of secreted protease variants exhibiting altered substrate specificity is a challenging task because effective screening methods for the desired property are not available yet. In this study, we sought to obtain variants of Kex2, a yeast Golgi protease, which exhibit altered P2 specificity. We first randomly mutated three Asp residues (D176, D210, and D211) that constitute the S2 pocket of Kex2 and then isolated from the resulting library Kex2 variants that preferred substrates with Met (poorly preferred by wild type Kex2) at the P2 position using a yeast-based screening method. The Kex2 variants isolated from this initial screening were further tested against various substrate sequences. Four out of the 16 isolated Kex2 variants showed greater preference for Met than for Lys (preferred by the wild-type Kex2) at the P2 position. We therefore suggest that our method might serve as an efficient tool for engineering and directing the evolution of secreted proteases.

Amino Acid Sequence↗
PubMed2004

Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.

Bacteria↗
PubMed2006

Evo-devo and constraints on selection.

Developmental bias, or genetic channeling, can influence the tempo and direction of evolution and, thus, become reflected in patterns of biodiversity. Twenty years ago, this notion rested on armchair descriptions of potential constraints on evolution. Now, a broad evo-devo approach involving both evolutionary and developmental genetics provides experimental analysis of such bias, revealing how shared genetic or developmental pathways among morphological traits contribute to the evolution of complexity and diversity, and that developmental bias itself evolves, generating variation in evolvability. As I discuss here, it is now possible to determine the relative importance of natural selection and of genetical and developmental architecture as determinants of organic diversity, particularly for examples of adaptive radiations and parallel evolution.

Animals↗
PubMed2005

Parallel capillary electrophoresis for the quantitative screening of fermentation broths containing natural products.

Directed molecular evolution is a recursive process of controlled genetic diversification and functional screening. The success of this approach is dependent on both the quality of the genetic diversity and the ability to accurately screen a large population of individual genetic variants for those having improved function. In this paper, the application of parallel capillary electrophoresis to rapidly quantitate lovastatin production levels by Aspergillus terreus mutants is described. A parallel 96 capillary instrument analyzed 900 samples in 8 h. with a 100 mM MES at pH 5.2 running buffer. In this manner, the fermentation broths of thousands of mutated strains were efficiently and inexpensively screened for increased lovastatin production. The ability to develop high-throughput methods to both separate and quantitate the components of complex mixtures greatly facilitates the ability to apply evolutionary engineering methods to complex biological systems.

Aspergillus↗
PubMed1999

Developmental regulation of dendritic cell function.

1998 saw key advances in our understanding of the molecular mechanisms whereby immature dendritic cells recognise foreign pathogens in tissues and are induced to migrate to secondary lymphoid organs. In particular, there have been some key insights into how dendritic cells subsequently direct the evolution of immune responses by differential expression of co-stimulatory molecules.

Animals↗
PubMed1991

Perspectives on the development of anti-HIV vaccines.

Progress towards the development of a vaccine against acquired immune deficiency syndrome is proceeding along several fronts. First and foremost, it rests on the basic research being done with the virus, particularly its mechanisms of replication, pathogenesis and evolution. More directly, progress comes from studies of animal models with the simian and human immunodeficiency viruses where vaccine candidates have proven effective in blocking infection. Principally because the animal models cannot answer all of the critical questions that apply to a vaccine for man, parallel studies in human volunteers have been initiated.

AIDS Vaccines↗
PubMed1997

Applications of DNA shuffling to pharmaceuticals and vaccines.

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.

Amino Acid Sequence↗
PubMed1996

Modeling protein-ligand complexes.

Increasing the rate at which new biologically active compounds are found is a major goal in pharmaceutical chemistry. Recently, several computational methods have been proposed with this intent. For some time, algorithms have been used to direct ligand evolution on the basis of complementarity to the three-dimensional structure of a selected protein. Current research focuses on enhancements to methods for searching chemical databases, proposing sensible modifications to known active compounds, and construction of novel ligands from theoretical principles.

Computer Simulation↗
PubMed1997

Variation in mate choice and mating preferences: a review of causes and consequences.

The aim of this review is to consider variation in mating preferences among females. We define mating preferences as the sensory and behavioural properties that influence the propensity of individuals to mate with certain phenotypes. Two properties of mating preferences can be distinguished: (1) "preference functions'-the order with which an individual ranks prospective mates and (2) "choosiness'-the effort an individual is prepared to invest in mate assessment. Patterns of mate choices can be altered by changing the costs of choosiness without altering the preference function. We discuss why it is important to study variation in female mating behaviour and identify five main areas of interest: Variation in mating preferences and costs of choosiness could (1) influence the rate and direction of evolution by sexual selection, (2) provide information about the evolutionary history of female preferences, (3) help explain inter-specific differences in the evolution of secondary sexual characteristics, (4) provide information about the level of benefits gained from mate choice, (5) provide information about the underlying mechanisms of mate choice. Variation in mate choice could be due to variability in preference functions, degree of choosiness, or both, and may arise due to genetic differences, developmental trajectories or proximate environmental factors. We review the evidence for genetic variation from genetic studies of heritability and also from data on the repeatability of mate-choice decisions (which can provide information about the upper limits to heritability). There can be problems in interpreting patterns of mate choice in terms of variation in mating preferences and we illustrate two main points. First, some factors can lead to mate choice patterns that mimic heritable variation in preferences and secondly other factors may obscure heritable preferences. These factors are divided into three overlapping classes, environmental, social and the effect of the female phenotype. The environmental factors discussed include predation risk and the costs of sampling; the social factors discussed include the effect of male-male interactions as well as female competition. We review the literature which presents data on how females sample males and discuss the number of cues females use. We conclude that sexual-selection studies have paid far less attention to variation among females than to variation among males, and that there is still much to learn about how females choose males and why different females make different choices. We suggest a number of possible lines for future research.

Animal Communication↗
PubMedDate not supplied

Confirmation of primary active substances from high throughput screening of chemical and biological populations: a statistical approach and practical considerations.

Many biologically important substances are discovered through screening of relevant chemical or biological libraries. The ability to find the active substances ("hits") from any random collection is largely determined by the quality of the assay and screening conditions. When a large population is screened for a specific characteristic, each member of that population is usually tested only once. Errors in the measurements require additional follow-up tests to confirm that each hit from the primary screen is truly active. In this report, we present a statistical model system that predicts the reliability of hits from a primary test as affected by the error in the assay and the choice of the hit threshold (hit limit). The hit confirmation rate, as well as false positive (representing substances that initially fall above the hit limit but whose true activity are below the hit limit) and false negative (representing substances that initially fall below the hit limit but whose true activity are in fact greater than the hit limit) rates have been analyzed with this model by computational simulation. This model can also be used in screen validation and post-screening data analysis. The statistical analysis presented here has broad implications and is applicable to screening of any large population for any specific characteristic. Obvious applications include drug discovery, gene chip analysis, population biology, directed molecular evolution, biological panning, and combinatorial material sciences.

Biological Factors↗
PubMed1997

Genetic algorithm based system for patient scheduling in highly constrained situations.

In medicine and health care there are a lot of situations when patients have to be scheduled on different devices and/or with different physicians or therapists. It may concern preventive examinations, laboratory tests or convalescent therapies, therefore we are always looking for an optimal schedule that would result in finishing all the activities scheduled as soon as possible, with the least patient waiting time and maximum device utilization. Since patient scheduling is a highly complex problem, it is impossible to make a qualitative schedule by hand or even with exact heuristic methods. Therefore we developed a powerful automated scheduling method for highly constrained situations based on genetic algorithms and machine learning. In this paper we present the method, together with the whole process of schedule generation, the important parameters to direct the evolution and how the algorithm is guaranteed to produce only feasible solutions, not breaking any of the required constraints. We applied the described method to a problem of scheduling patients with different therapy needs to a limited number of therapeutic devices, but the algorithm can be easily modified for use in similar situations. The results are quite encouraging and since all the solutions are feasible, the method can be easily incorporated into an interactive user interface, which can be of major importance when scheduling patients, and human resources in general, is considered.

Algorithms↗
PubMed1997

The advantage of sex in evolving yeast populations.

Sex is a general feature of the life cycle of eukaryotes. It is not universal, however, as many organisms seem to lack sex entirely. The widespread occurrence of sex is puzzling, both because meiotic recombination can disrupt co-adapted combinations of genes, and because it halves the potential rate of reproduction in organisms with strongly differentiated male and female gametes. Most attempts to explain the maintenance of sexuality invoke differences between parents and sexual offspring. These differences may be advantageous in novel or changing environments if new gene combinations are favoured from time to time. Sex would then serve to concentrate beneficial mutations that have arisen independently into the same line of descent. But in a stable environment sex might serve to concentrate deleterious mutations, so that they will be more effectively purged from the population by selection. We have studied the effect of sex on mean fitness in experimental populations of the budding yeast Saccharomyces cerevisiae. Our results show that sex increases mean fitness in an environment to which the populations were well adapted, but not in an environment to which new adaptation occurred, supporting the hypothesis that the advantage of sexuality lay in the removal of deleterious mutations.

Adaptation, Physiological↗
PubMed1999

A metal complex that binds alpha-amino acids with high and predictable stereospecificity.

Molecular recognition is the key step in a wide range of controlled separation and chemical transformation processes, with enzymes performing this task with an unsurpassed degree of selectivity. Enzymes contain only 20 simple amino acids, yet it remains difficult to rationalize or even predict these stereospecific recognition events. Nonetheless, the rational design of receptors able to recognize amino acids stereospecifically is attracting considerable interest because therapeutic drugs, that may be developed from chiral amino acid intermediates, are increasingly required in enantiomerically pure form. Early work has stimulated the development of efficient receptors based on small molecules, but binding of amino acids with high and predictable stereospecificity remains difficult to achieve. Directed molecular evolution, on the other hand, does select for RNA sequences or antibodies that bind amino acids with high specificity, but typically without providing insights into the molecular recognition mechanisms involved. Here we show that a rationally designed metal complex formed from a trivalent cobalt ion and a tetradentate ligand binds natural amino acids, including the simple yet challenging amino acid alanine, with high and predictable regio- and stereospecificity. We expect that our approach will allow the binding as well as separation and stereospecific catalytic formation of its target amino acids.

Amino Acids↗
PubMed2004

The 'evolvability' of promiscuous protein functions.

How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level? Results from directed laboratory evolution experiments indicate that the evolution of a new function is driven by mutations that have little effect on the native function but large effects on the promiscuous functions that serve as starting point. Thus, an evolving protein can initially acquire increased fitness for a new function without losing its original function. Gene duplication and the divergence of a completely new protein may then follow.

Aryldialkylphosphatase↗
PubMed2004

Structural visualization of polarization fatigue in epitaxial ferroelectric oxide devices.

Ferroelectric oxides, such as Pb(Zr,Ti)O(3), are useful for electronic and photonic devices because of their ability to retain two stable polarization states, which can form the basis for memory and logic circuitry. Requirements for long-term operation of practical devices such as non-volatile RAM (random access memory) include consistent polarization switching over many (more than 10(12)) cycles of the applied electric field, which represents a major challenge. As switching is largely controlled by the motion and pinning of domain walls, it is necessary to develop suitable tools that can directly probe the ferroelectric domain structures in operating devices-thin-film structures with electrical contacts. A recently developed synchrotron X-ray microdiffraction technique complements existing microscopic probes, and allows us to visualize directly the evolution of polarization domains in ferroelectric devices, through metal or oxide electrodes, and with submicrometre spatial resolution. The images reveal two regimes of fatigue, depending on the magnitude of the electric field pulses driving the device: a low-field regime in which fatigue can be reversed with higher electric field pulses, and a regime at very high electric fields in which there is a non-reversible crystallographic relaxation of the epitaxial ferroelectric film.

Journal Article↗
PubMed2006

Functional dependency of structures of ionic liquids: do substituents govern the selectivity of enzymatic glycerolysis?

The concept of regulating the preference of a reversible multi-step reaction by adjusting the substituents of ionic liquids (ILs) has been successfully exemplified with a group of tetraammonium-based ionic liquids as medium for the enzymatic glycerolysis. Simultaneous existence of long chain hydrophobic substituents and hydrophilic ethoxyl or hydroxyl moieties is found, respectively, to be essential for triglycerides (TG) dissolving and equilibrium shifting. The reactions in the ILs with cations consisting of long chain and free hydroxyl groups gave markedly higher conversion of TG and better preference to monoglyceride formation. Interestingly the predicted results from COSMO-RS (a quantum chemical model programme) achieved a good agreement with the experimental data, mapping out the specific solvation from the ILs as well as demonstrating the interaction between ILs, substrates and products being the intrinsic causes that govern reaction evolution and direct equilibrium shifting.

Glycerol↗