The Experts below are selected from a list of 741 Experts worldwide ranked by ideXlab platform

David A. Mcclellan - One of the best experts on this subject based on the ideXlab platform.

  • physicochemical evolution and molecular adaptation of the cetacean and artiodactyl cytochrome b proteins
    Molecular Biology and Evolution, 2005
    Co-Authors: David A. Mcclellan, E J Palfreyman, M J Smith, J L Moss, R G Christensen, Joshua Sailsbery
    Abstract:

    : Cetaceans have most likely experienced metabolic shifts since evolutionarily diverging from their terrestrial ancestors, shifts that may be reflected in the proteins such as cytochrome b that are responsible for metabolic efficiency. However, accepted statistical methods for detecting molecular adaptation are largely biased against even moderately conservative proteins because the primary criterion involves a comparison of nonsynonymous and synonymous substitution rates (dN/dS); they do not allow for the possibility that adaptation may come in the form of very few amino acid changes. We apply the MM01 model to the possible molecular adaptation of cytochrome b among cetaceans because it does not rely on a dN/dS ratio, instead evaluating positive selection in terms of the amino acid properties that comprise protein phenotypes that selection at the molecular level may act upon. We also apply the Codon-Degeneracy model (CDM), which focuses on evaluating overall patterns of nucleotide substitution in terms of base exchange, Codon position, and synonymy to estimate the overall effect of selection. Using these relatively new models, we characterize the molecular adaptation that has occurred in the cetacean cytochrome b protein by comparing revealed amino acid replacement patterns to those found among artiodactyls, the modern terrestrial mammals found to be most closely related to cetaceans. Our findings suggest that several regions of the cetacean cytochrome b protein have experienced molecular adaptation. Also, these adaptations are spatially associated with domain structure, protein function, and the structure and function of the cytochrome bc(1) complex and its constituents. We also have found a general correlation between the results of the analytical software programs TreeSAAP (which implements the MM01 model) and CDM (which implements the Codon-Degeneracy model).

  • the phylogenetic utility of the Codon Degeneracy model
    Journal of Molecular Evolution, 2000
    Co-Authors: David A. Mcclellan
    Abstract:

    The Codon-Degeneracy model (CDM) predicts relative frequencies of substitution for any set of homologous protein-coding DNA sequences based on patterns of nucleotide Degeneracy, Codon composition, and the assumption of selective neutrality. However, at present, the CDM is reliant on outside estimates of transition bias. A new method by which the power of the CDM can be used to find a synonymous transition bias that is optimal for any given phylogenetic tree topology is presented. An example is illustrated that utilizes optimized transition biases to generate CDM GF-scores for every possible phylogenetic tree for pocket gophers of the genus Orthogeomys. The resulting distribution of CDM GF-scores is compared and contrasted with the results of maximum parsimony and maximum likelihood methods. Although convergence on a single tree topology by the CDM and another method indicates greater support for that particular tree, the value of CDM GF-score as the sole optimality criterion for phylogeny reconstruction remains to be determined. It is clear, however, that the a priori estimation of an optimum transition bias from Codon composition has a direct application to differentiating between alternative trees.

  • The Codon-Degeneracy Model of Molecular Evolution
    Journal of molecular evolution, 2000
    Co-Authors: David A. Mcclellan
    Abstract:

    Mitochondrial genetic Codons can be categorized by four patterns of nucleotide-site Degeneracy based on varying combinations of twofold- or nondegenerate sites at first Codon positions and twofold- or fourfold-degenerate sites at third Codon positions. Herein, a model of molecular evolution is introduced that uses these patterns to calculate expected substitution frequencies for each Codon position and substitution type relative to overall number of synonymous or nonsynonymous substitutions. Regions of the pocket gopher cytochrome oxidase subunit I (COI) and cytochrome b (cyt-b) genes are analyzed using this model. Chi-square distributions are used to produce relative goodness-of-fit (GF) scores for measuring the difference between substitution frequencies predicted by the Codon-Degeneracy model (CDM), and frequencies inferred using a well-supported phylogenetic tree of closely related species. The GF scores for expected and observed synonymous (GF(syn) = 0.429, p = 0.807) and nonsynonymous (GF(ns) = 2.309, p = 0.679) substitution frequencies resulted in a failure to reject the CDM as a null hypothesis for the molecular evolution of COI and cyt-b in pocket gophers. Alternative tree topologies and calculations of transition bias for these data result in higher GF scores.

Manfred T Reetz - One of the best experts on this subject based on the ideXlab platform.

  • structure guided triple code saturation mutagenesis efficient tuning of the stereoselectivity of an epoxide hydrolase
    ACS Catalysis, 2016
    Co-Authors: Zhoutong Sun, Richard Lonsdale, Jianbo Wang, Jiahai Zhou, Manfred T Reetz
    Abstract:

    The directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnology—the often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK Codon Degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 1015 transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200–800 transformants in one library. The triple c...

  • Structure-Guided Triple-Code Saturation Mutagenesis: Efficient Tuning of the Stereoselectivity of an Epoxide Hydrolase
    2016
    Co-Authors: Zhoutong Sun, Richard Lonsdale, Jianbo Wang, Jiahai Zhou, Manfred T Reetz
    Abstract:

    The directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnologythe often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK Codon Degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 1015 transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200–800 transformants in one library. The triple code utilizes structural (X-ray) and consensus-derived sequence data, and is therefore designed to match the steric and electrostatic characteristics of the particular enzyme. Using this approach, limonene epoxide hydrolase has been successfully engineered as stereoselective catalysts in the hydrolytic desymmetrization of meso-type epoxides with formation of either (R,R)- or (S,S)-configurated diols on an optional basis and kinetic resolution of chiral substrates. Crystal structures and docking computations support the source of notably enhanced and inverted enantioselectivity

  • addressing the numbers problem in directed evolution
    ChemBioChem, 2008
    Co-Authors: Manfred T Reetz, Daniel Kahakeaw, Renate Lohmer
    Abstract:

    Our previous contribution to increasing the efficiency of directed evolution is iterative saturation mutagenesis (ISM) as a systematic means of generating focused libraries for the control of substrate acceptance, enantioselectivity, or thermostability of enzymes. We have now introduced a crucial element to knowledge-guided targeted mutagenesis in general that helps to solve the numbers problem in directed evolution. We show that the choice of the amino acid (aa) alphabet, as specified by the utilized Codon Degeneracy, provides the experimenter with a powerful tool in designing "smarter" randomized libraries that require considerably less screening effort. A systematic comparison of two different Codon degeneracies was made by examining the relative quality of the identically sized enzyme libraries in relation to the degree of oversampling required in the screening process. The specific example in our case study concerns the conventional NNK Codon Degeneracy (32 Codons/20 aa) versus NDT (12 Codons/12 aa). The model reaction is the hydrolytic kinetic resolution of a chiral trans-disubstituted epoxide, catalyzed by the epoxide hydrolase from Aspergillus niger. The NDT library proves to be of much higher quality, as measured by the dramatically higher frequency of positive variants and by the magnitude of catalyst improvement (enhanced rate and enantioselectivity). We provide a statistical analysis that constitutes a useful guide for the optimal design and generation of "smarter" focused libraries. This type of approach accelerates the process of laboratory evolution considerably and can be expected to be broadly applicable when engineering functional proteins in general.

R.h. Duncan Lyngdoh - One of the best experts on this subject based on the ideXlab platform.

  • Configuration of wobble base pairs having pyrimidines as antiCodon wobble bases: significance for Codon Degeneracy
    2014
    Co-Authors: Gunajyoti Das, R.h. Duncan Lyngdoh
    Abstract:

    Degeneracy of the genetic code was attributed by Crick to imprecise hydrogen-bonded base-pairing at the wobble position during Codon–antiCodon pairing. The Crick wobble rules define but do not explain the RNA base pair combinations allowed at this position. We select six pyrimidine bases functioning as antiCodon wobble bases (AWBs) to study their H-bonded pairing properties with the four major RNA bases using density functional theory at the B3LYP/6-31G(d,p) level. This is done to assess the extent to which the configuration of a solitary RNA wobble base pair may in itself determine specificity and Degeneracy of the genetic code by allowing or disallowing the given base pair during Codon–antiCodon pairing. Calculated values of select configuration markers for the base pairs screen well between allowed and disallowed base pairs for most cases examined here, where the base pair width emerges as an important factor. A few allowed wobble pairs invoke the involvement of RNA nucleoside conformation, as well as involvement of the exocyclic substituent in H-bonding. This study, however, cannot explain the disallowed status of the Ura⋯Gua wobble pair on the basis of configuration alone. Explanation of the allowed status of the V⋯Ura pair requires further study on the mediatory role of water molecules. Apart from these two cases, these computational results are sufficient, on the basis of base pair configuration alone, to account for the specificity and Degeneracy of the genetic code for all known cases of Codon–antiCodon pairing which involve the pyrimidine AWBs studied here.

  • Configuration of wobble base pairs having pyrimidines as antiCodon wobble bases: significance for Codon Degeneracy.
    Journal of biomolecular structure & dynamics, 2013
    Co-Authors: Gunajyoti Das, R.h. Duncan Lyngdoh
    Abstract:

    Degeneracy of the genetic code was attributed by Crick to imprecise hydrogen-bonded base-pairing at the wobble position during Codon–antiCodon pairing. The Crick wobble rules define but do not explain the RNA base pair combinations allowed at this position. We select six pyrimidine bases functioning as antiCodon wobble bases (AWBs) to study their H-bonded pairing properties with the four major RNA bases using density functional theory at the B3LYP/6-31G(d,p) level. This is done to assess the extent to which the configuration of a solitary RNA wobble base pair may in itself determine specificity and Degeneracy of the genetic code by allowing or disallowing the given base pair during Codon–antiCodon pairing. Calculated values of select configuration markers for the base pairs screen well between allowed and disallowed base pairs for most cases examined here, where the base pair width emerges as an important factor. A few allowed wobble pairs invoke the involvement of RNA nucleoside conformation, as well as ...

  • Role of wobble base pair geometry for Codon Degeneracy: purine-type bases at the antiCodon wobble position
    Journal of molecular modeling, 2012
    Co-Authors: Gunajyoti Das, R.h. Duncan Lyngdoh
    Abstract:

    Codon Degeneracy is a key feature of the genetic code, explained by Crick (J Mol Biol 19:548-555, 1966) in terms of imprecision of base pairing at the Codon third position (the wobble position) of the Codon-antiCodon duplex. The Crick wobble rules define, but do not explain, which base pairs are allowed/disallowed at the wobble position of this duplex. This work examines whether the H-bonded configurations of solitary RNA base pairs can in themselves help decide which base pairs are allowed at the wobble position during Codon-antiCodon pairing. Taking the purine-type bases guanine, hypoxanthine, queuine and adenine as antiCodon wobble bases, H-bonded pairing energies and optimized configurations of numerous RNA base pairs are calculated in gas and modeled aqueous phase at the B3LYP/6-31 G(d,p) level. Calculated descriptors of alignment of these solitary base pairs are able to screen between allowed and disallowed base pairs for all cases studied here, except two cases which invoke base-sugar interactions in the Codon wobble nucleoside. The exclusion of adenine from the antiCodon wobble position cannot be explained on the basis of pairing facility or base pair geometry. These DFT results thus account for the specificity and Degeneracy of the genetic code for all cases involving guanine, hypoxanthine and queuine as antiCodon wobble bases.

T. Martin Embley - One of the best experts on this subject based on the ideXlab platform.

  • Compositional biases among synonymous substitutions cause conflict between gene and protein trees for plastid origins.
    Molecular Biology and Evolution, 2014
    Co-Authors: Blaise Li, Joao S Lopes, Peter G Foster, T. Martin Embley
    Abstract:

    Archaeplastida (=Kingdom Plantae) are primary plastid-bearing organisms that evolved via the endosymbiotic association of a heterotrophic eukaryote host cell and a cyanobacterial endosymbiont approximately 1,400 Ma. Here, we present analyses of cyanobacterial and plastid genomes that show strongly conflicting phylogenies based on 75 plastid (or nuclear plastid-targeted) protein-coding genes and their direct translations to proteins. The conflict between genes and proteins is largely robust to the use of sophisticated data- and tree-heterogeneous composition models. However, by using nucleotide ambiguity codes to eliminate synonymous substitutions due to Codon-Degeneracy, we identify a composition bias, and dependent Codon-usage bias, resulting from synonymous substitutions at all third Codon positions and first Codon positions of leucine and arginine, as the main cause for the conflicting phylogenetic signals. We argue that the protein-coding gene data analyses are likely misleading due to artifacts induced by convergent composition biases at first Codon positions of leucine and arginine and at all third Codon positions. Our analyses corroborate previous studies based on gene sequence analysis that suggest Cyanobacteria evolved by the early paraphyletic splitting of Gloeobacter and a specific Synechococcus strain (JA33Ab), with all other remaining cyanobacterial groups, including both unicellular and filamentous species, forming the sister-group to the Archaeplastida lineage. In addition, our analyses using better-fitting models suggest (but without statistically strong support) an early divergence of Glaucophyta within Archaeplastida, with the Rhodophyta (red algae), and Viridiplantae (green algae and land plants) forming a separate lineage.

  • Compositional Biases among Synonymous Substitutions Cause Conflict between Gene and Protein Trees for Plastid Origins
    'Oxford University Press (OUP)', 2011
    Co-Authors: Li B, T. Martin Embley, Js Lopes, Pg Foster, Cj Cox
    Abstract:

    Archaeplastida (=Kingdom Plantae) are primary plastid-bearing organisms that evolved via the endosymbiotic association of a heterotrophic eukaryote host cell and a cyanobacterial endosymbiont approximately 1,400 Ma. Here, we present analyses of cyanobacterial and plastid genomes that show strongly conflicting phylogenies based on 75 plastid (or nuclear plastid-targeted) protein-coding genes and their direct translations to proteins. The conflict between genes and proteins is largely robust to the use of sophisticated data- and tree-heterogeneous composition models. However, by using nucleotide ambiguity codes to eliminate synonymous substitutions due to Codon-Degeneracy, we identify a composition bias, and dependent Codon-usage bias, resulting from synonymous substitutions at all third Codon positions and first Codon positions of leucine and arginine, as the main cause for the conflicting phylogenetic signals. We argue that the protein-coding gene data analyses are likely misleading due to artifacts induced by convergent composition biases at first Codon positions of leucine and arginine and at all third Codon positions. Our analyses corroborate previous studies based on gene sequence analysis that suggest Cyanobacteria evolved by the early paraphyletic splitting of Gloeobacter and a specific Synechococcus strain (JA33Ab), with all other remaining cyanobacterial groups, including both unicellular and filamentous species, forming the sister-group to the Archaeplastida lineage. In addition, our analyses using better-fitting models suggest (but without statistically strong support) an early divergence of Glaucophyta within Archaeplastida, with the Rhodophyta (red algae), and Viridiplantae (green algae and land plants) forming a separate lineage.Copyright The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. The attached file is the published version of the article

Paola Arlotta - One of the best experts on this subject based on the ideXlab platform.

  • Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription
    Nature biotechnology, 2011
    Co-Authors: Feng Zhang, Le Cong, Simona Lodato, Sriram Kosuri, George M. Church, Paola Arlotta
    Abstract:

    The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator-like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage Codon Degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.