Adaptive evolution of metabolic pathways in Drosophila

The adaptive significance of enzyme variation has been of central interest in population genetics. Yet, how natural selection operates on enzymes in the larger context of biochemical pathways has not been broadly explored. A basic expectation is that natural selection on metabolic phenotypes will target enzymes that control metabolic flux, but how adaptive variation is distributed among enzymes in metabolic networks is poorly understood. Here, we use population genetic methods to identify enzymes responding to adaptive selection in the pathways of central metabolism in Drosophila melanogaster and Drosophila simulans. We report polymorphism and divergence data for 17 genes that encode enzymes of 5 metabolic pathways that converge at glucose-6-phosphate (G6P). Deviations from neutral expectations were observed at five loci. Of the 10 genes that encode the enzymes of glycolysis, only aldolase (Ald) deviated from neutrality. The other 4 genes that were inconsistent with neutral evolution (glucose-6-phosphate dehydrogenase [G6pd]), phosphoglucomutase [Pgm], trehalose-6-phosphate synthetase [Tps1], and glucose-6phosphatase [G6pase] encode G6P branch point enzymes that catalyze reactions at the entry point to the pentose-phosphate, glycogenic, trehalose synthesis, and gluconeogenic pathways. We reconcile these results with population genetics theory and existing arguments on metabolic regulation and propose that the incidence of adaptive selection in this system is related to the distribution of flux control. The data suggest that adaptive evolution of G6P branch point enzymes may have special significance in metabolic adaptation.     

Right-handed snakes: convergent evolution of asymmetry for functional specialization

External asymmetry found in diverse animals bears critical functions to fulfil ecological requirements. Some snail-eating arthropods exhibit directional asymmetry in their feeding apparatus for foraging efficiency because dextral (clockwise) species are overwhelmingly predominant in snails. Here, we show convergence of directional asymmetry in the dentition of snail-eating vertebrates. We found that snakes in the subfamily Pareatinae, except for non-snail-eating specialists, have more teeth on the right mandible than the left. In feeding experiments, a snail-eating specialist Pareas iwasakii completed extracting a dextral soft body faster with fewer mandible retractions than a sinistral body. The snakes failed in holding and dropped sinistral snails more often owing to behavioural asymmetry when striking. Our results demonstrate that symmetry break in dentition is a key innovation that has opened a unique ecological niche for snake predators.     

The evolution of core proteins involved in microRNA biogenesis

Background  

MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs (ncRNAs) which play important roles in eukaryotic gene regulation. miRNA biogenesis and activation is a complex process involving multiple protein catalysts and involves the large macromolecular RNAi Silencing Complex or RISC. While phylogenetic analyses of miRNA genes have been previously published, the evolution of miRNA biogenesis itself has been little studied. In order to better understand the origin of miRNA processing in animals and plants, we determined the phyletic occurrences and evolutionary relationships of four major miRNA pathway protein components; Dicer, Argonaute, RISC RNA-binding proteins, and Exportin-5.     

Non-divergence theory of evolution: sequence comparison of some proteins from snakes and bacteria

A "non-divergence theory" is proposed for the mechanism of evolution. The theory is based on the observation that comparison of the amino acid sequences of related proteins in various organisms gives inconsistent results from one type of protein to another, and on the occurrence of significant gene transfer among living organisms. Special attention is focused on the sequence comparisons of short- and long-chain neurotoxins and phospholipases A2 from the venoms of proteroglyphous snakes and those of microbial ferredoxins, rubredoxins, and flavodoxins.     

Molecular evolution of the infrared sensory gene TRPA1 in snakes and implications for functional studies

TRPA1 is a calcium ion channel protein recently identified as the infrared receptor in pit organ-containing snakes. Therefore, understanding the molecular evolution of TRPA1 may help to illuminate the origin of "heat vision" in snakes and reveal the molecular mechanism of infrared sensitivity for TRPA1. To this end, we sequenced the infrared sensory gene TRPA1 in 24 snake species, representing nine snake families and multiple non-snake outgroups. We found that TRPA1 is under strong positive selection in the pit-bearing snakes studied, but not in other non-pit snakes and non-snake vertebrates. As a comparison, TRPV1, a gene closely related to TRPA1, was found to be under strong purifying selection in all the species studied, with no difference in the strength of selection between pit-bearing snakes and non-pit snakes. This finding demonstrates that the adaptive evolution of TRPA1 specifically occurred within the pit-bearing snakes and may be related to the functional modification for detecting infrared radiation. In addition, by comparing the TRPA1 protein sequences, we identified 11 amino acid sites that were diverged in pit-bearing snakes but conserved in non-pit snakes and other vertebrates, 21 sites that were diverged only within pit-vipers but conserved in the remaining snakes. These specific amino acid substitutions may be potentially functional important for infrared sensing.     

Structural and functional evolution of transthyretin and transthyretin-like proteins

Transthyretin (TTR) is a tetrameric protein involved in the distribution of thyroid hormones in vertebrates. The amino acid sequence of TTR is highly conserved across vertebrates. Hypothetical TTR-like proteins (TLPs) were inferred from the identification of genes in nonvertebrate species. Here, we identified five motifs defining TLPs and three motifs defining both TTRs and TLPs. These motifs were mapped onto structurally conserved and functionally important regions of TTRs. These motifs were used to build hidden Markov models for accurate identification of TLPs in other organisms. TLPs were divided into three main groups based on their N-terminal regions. Most TLPs are cytosolic, but in plants and slime mold, we predict they are peroxisomal. We verified that the TLPs from enterobacteria were periplasmic. We demonstrated that TLP genes are expressed in a bacterium (E. coli), an invertebrate animal (C. elegans), and a plant (A. thaliana). These TLPs have similar subunit molecular weights to TTRs, are tetramers, and are predicted to have similar three-dimensional (3D) structures to TTRs, but do not bind thyroid hormones or similar ligands. We suggest that like TTRs, the N-terminal and C-terminal regions of TLPs are integral in defining the function of TLPs in nonvertebrate species and that the TLP gene duplicated in primitive vertebrates to produce the TTR gene. TLP/TTR has retained its overall structure, but changed function and localization during evolution in bacteria, invertebrates, plants, and vertebrates.     

Understanding relationship between sequence and functional evolution in yeast proteins

The underlying relationship between functional variables and sequence evolutionary rates is often assessed by partial correlation analysis. However, this strategy is impeded by the difficulty of conducting meaningful statistical analysis using noisy biological data. A recent study suggested that the partial correlation analysis is misleading when data is noisy and that the principal component regression analysis is a better tool to analyze biological data. In this paper, we evaluate how these two statistical tools (partial correlation and principal component regression) perform when data are noisy. Contrary to the earlier conclusion, we found that these two tools perform comparably in most cases. Furthermore, when there is more than one ‘true’ independent variable, partial correlation analysis delivers a better representation of the data. Employing both tools may provide a more complete and complementary representation of the real data. In this light, and with new analyses, we suggest that protein length and gene dispensability play significant, independent roles in yeast protein evolution. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The evolution of venom-delivery systems in snakes

The Colubroidea represents approximately 2300 of the 2700 species of living snakes and includes all venomous taxa. Although many morphological studies of colubroid snakes have been carried over the last hundred years, the phylogenetic relationships within this group are poorly known. In this study, components of the venom-delivery system (VDS) were examined within the context of two conflicting phylogenetic hypotheses proposed in 1988 by Cadle and in 1998 by Kraus & Braun. The results suggest that several major morphological changes occurred early in colubroid evolution: a Duvernoy's gland evolved, the posterior maxillary teeth became specialized relative to the anterior maxillary teeth, and the attachment of the pterygoideus muscle moved forward to a position associated with the posterior maxillary teeth. These innovations may have allowed the great radiation of colubroid snakes that led to the Colubroidea representing such a large percentage of living snakes. More recently, three separate lineages of colubroids have independently evolved highly specialized front-fanged VDSs with large and complex venom glands, venom gland compressor muscles, and tubular fangs.  © 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137 , 337−354.     

Gravity and the evolution of cardiopulmonary morphology in snakes

Physiological investigations of snakes have established the importance of heart position and pulmonary structure in contexts of gravity effects on blood circulation. Here we investigate morphological correlates of cardiopulmonary physiology in contexts related to ecology, behavior and evolution. We analyze data for heart position and length of vascular lung in 154 species of snakes that exhibit a broad range of characteristic behaviors and habitat associations. We construct a composite phylogeny for these species, and we codify gravitational stress according to species habitat and behavior. We use conventional regression and phylogenetically independent contrasts to evaluate whether trait diversity is correlated with gravitational habitat related to evolutionary transitions within the composite tree topology. We demonstrate that snake species living in arboreal habitats, or which express strongly climbing behaviors, possess relatively short blood columns between the heart and the head, as well as relatively short vascular lungs, compared to terrestrial species. Aquatic species, which experience little or no gravity stress in water, show the reverse — significantly longer heart-head distance and longer vascular lungs. These phylogenetic differences complement the results of physiological studies and are reflected in multiple habitat transitions during the evolutionary histories of these snake lineages, providing strong evidence that heart-to-head distance and length of vascular lung are co-adaptive cardiopulmonary features of snakes.     

Tracheal diverticula in snakes: possible functions and evolution

The structural features of tracheal diverticula are reviewed, and their taxonomic distribution summarized. Five possible functions are hypothesized for these structures: as an air reservoir, as an accessory site for gas exchange, to enhance buoyancy, to inflate the neck, and to modify the hiss acoustically. Three possible scenarios for the evolutionary origin of tracheal diverticula are discussed: through simplification of tracheal lungs, through subdivision of expanded tracheal membranes, and as de novo structures. The tracheal diverticula most likely function concomitantly to inflate the neck and acoustically modify the hiss. These structures presumably arose de novo , in either one or two evolutionary lineages.     

Modularity of the hydrophobic core and evolution of functional diversity in fold A glycosyltransferases

Hydrophobic cores are fundamental structural properties of proteins typically associated with protein folding and stability; however, how the hydrophobic core shapes protein evolution and function is poorly understood. Here, we investigated the role of conserved hydrophobic cores in fold-A glycosyltransferases (GT-As), a large superfamily of enzymes that catalyze formation of glycosidic linkages between diverse donor and acceptor substrates through distinct catalytic mechanisms (inverting versus retaining). Using hidden Markov models and protein structural alignments, we identify similarities in the phosphate-binding cassette (PBC) of GT-As and unrelated nucleotide-binding proteins, such as UDP-sugar pyrophosphorylases. We demonstrate that GT-As have diverged from other nucleotide-binding proteins through structural elaboration of the PBC and its unique hydrophobic tethering to the F-helix, which harbors the catalytic base (xED-Asp). While the hydrophobic tethering is conserved across diverse GT-A fold enzymes, some families, such as B3GNT2, display variations in tethering interactions and core packing. We evaluated the structural and functional impact of these core variations through experimental mutational analysis and molecular dynamics simulations and find that some of the core mutations (T336I in B3GNT2) increase catalytic efficiency by modulating the conformational occupancy of the catalytic base between “D-in” and acceptor-accessible “D-out” conformation. Taken together, our studies support a model of evolution in which the GT-A core evolved progressively through elaboration upon an ancient PBC found in diverse nucleotide-binding proteins, and malleability of this core provided the structural framework for evolving new catalytic and substrate-binding functions in extant GT-A fold enzymes.     

New genotype-phenotype linkages for directed evolution of functional proteins

The key practical consideration in directed evolution of functional biomolecules is the linkage of genotype and phenotype. In vitro selections offer the potential to select from libraries with up to 10(10)-10(14) members, with fewer constraints than current cell-based selections. New approaches such as mRNA display, ribosome display and in vitro compartmentalisation have complementary areas of application in selections for binding or catalysis.     

Adaptive evolution in mammalian proteins involved in cochlear outer hair cell electromotility

Somatic electromotility in cochlear outer hair cells, as the basis for cochlear amplification, is a mammalian novelty and it is largely dependent upon rapid cell length changes proposed to be mediated by the motor-protein prestin, a member of the solute carrier anion-transport family 26. Thus, one might predict that prestin has specifically evolved in mammals to support this unique mammalian adaptation. Using codon-based likelihood models we found evidences for positive selection in the motor-protein prestin only in the mammalian lineage, supporting the hypothesis that lineage-specific adaptation-driven molecular changes endowed prestin with the ability to mediate somatic electromotility. Moreover, signatures of positive selection were found on the alpha10, but not the alpha9, nicotinic cholinergic receptor subunits. An alpha9alpha10-containing nicotinic cholinergic receptor mediates inhibitory olivocochlear efferent effects on hair cells across vertebrates. Our results suggest that evolution-driven modifications of the alpha10 subunit probably allowed the alpha9alpha10 heteromeric receptor to serve a differential function in the mammalian cochlea. Thus, we describe for the first time at the molecular level signatures of adaptive evolution in two outer hair cell proteins only in the lineage leading to mammals. This finding is most likely related with the roles these proteins play in somatic electromotility and/or its fine tuning.     

Identification of residues in glutathione transferase capable of driving functional diversification in evolution. A novel approach to protein redesign

Evolution of protein function can be driven by positive selection of advantageous nonsynonymous codon mutations that arise following gene duplication. By observing the presence and degree of site-specific positive selection for change between divergent paralogs, residue positions responsible for functional changes can be identified. We applied this analysis to genes encoding Mu class glutathione transferases, which differ widely in substrate specificities. Approximately 3% of the amino acid residue positions, both near to and distant from the active site, are under statistically significant positive selection for change. Relevant human glutathione transferase (GST) M1-1 and GST M2-2 codons were mutated. A chemically conservative threonine to serine mutation in GST M2-2 elicited a 1,000-fold increase in specific activity with the GST M1-1-specific substrate trans-stilbene oxide and a 30-fold increase with the alternative epoxide substrates styrene oxide and nitrophenyl glycidol. The reverse mutation in GST M1-1 resulted in reciprocal decreases in activity. Thus, identification of hypervariable codon positions can be a powerful aid in the redesign of protein function, lessening the requirement for extensive mutagenesis or structural knowledge and sometimes suggesting mutations that would otherwise be considered functionally conservative.     

Employing directed evolution for the functional analysis of multi-specific proteins

Multi-specific proteins located at the heart of complex protein–protein interaction (PPI) networks play essential roles in the survival and fitness of the cell. In addition, multi-specific or promiscuous enzymes exhibit activity toward a wide range of substrates so as to increase cell evolvability and robustness. However, despite their high importance, investigating the in vivo function of these proteins is difficult, due to their complex nature. Typically, deletion of these proteins leads to the abolishment of large PPI networks, highlighting the difficulty in examining the contributions of specific interactions/activities to complex biological processes and cell phenotypes. Protein engineering approaches, including directed evolution and computational protein design, allow for the generation of multi-specific proteins in which certain activities remain intact while others are abolished. The generation and examination of these mutants both in vitro and in vivo can provide high-resolution analysis of biological processes and cell phenotypes and provide new insight into the evolution and molecular function of this important protein family.     

Adaptive evolution in locomotor performance: How selective pressures and functional relationships produce diversity

Despite the complexity of nature, most comparative studies of phenotypic evolution consider selective pressures in isolation. When competing pressures operate on the same system, it is commonly expected that trade‐offs will occur that will limit the evolution of phenotypic diversity, however, it is possible that interactions among selective pressures may promote diversity instead. We explored the evolution of locomotor performance in lizards in relation to possible selective pressures using the Ornstein–Uhlenbeck process. Here, we show that a combination of selection based on foraging mode and predator escape is required to explain variation in performance phenotypes. Surprisingly, habitat use contributed little explanatory power. We find that it is possible to evolve very different abilities in performance which were previously thought to be tightly correlated, supporting a growing literature that explores the many‐to‐one mapping of morphological design. Although we generally find the expected trade‐off between maximal exertion and speed, this relationship surprisingly disappears when species experience selection for both performance types. We conclude that functional integration need not limit adaptive potential, and that an integrative approach considering multiple major influences on a phenotype allows a more complete understanding of adaptation and the evolution of diversity.     

核糖体展示及体外分子选择与进化

核糖体展示是20世纪90年代中期发展起来的一种简便而有效的体外分子选择与进化技术。它也是第一种完全在体外进行蛋白质或多肽分子选择与进化的方法。本主要概述了体外核糖体展示技术的建立基础、基本原理和技术特点等,并跟踪了目前该领域的最新研究进展和发展前景。     

Adaptive evolution of class 5 fimbrial genes in enterotoxigenic Escherichia coli and its functional consequences

Class 5 fimbriae of enterotoxigenic Escherichia coli (ETEC) comprise eight serologically discrete colonization factors that mediate small intestinal adhesion. Their differentiation has been attributed to the pressure imposed by host adaptive immunity. We sequenced the major pilin and minor adhesin subunit genes of a geographically diverse population of ETEC elaborating CFA/I (n = 31), CS17 (n = 20), and CS2 (n = 18) and elucidated the functional effect of microevolutionary processes. Between the fimbrial types, the pairwise nucleotide diversity for the pilin or adhesin genes ranged from 35-43%. Within each fimbrial type, there were 17 non-synonymous and 1 synonymous point mutations among all pilin or adhesin gene copies, implying that each fimbrial type was acquired by ETEC strains very recently, consistent with a recent origin of this E. coli pathotype. The 17 non-synonymous allelic differences occurred in the CFA/I pilin gene cfaB (two changes) and adhesin gene cfaE (three changes), and CS17 adhesin gene csbD (12 changes). All but one amino acid change in the adhesins clustered around the predicted ligand-binding pocket. Functionally, these changes conferred an increase in cell adhesion in a flow chamber assay. In contrast, the two mutations in the non-adhesive CfaB subunit localized to the intersubunit interface and significantly reduced fimbrial adhesion in this assay. In conclusion, naturally occurring mutations in the ETEC adhesive and non-adhesive subunits altered function, were acquired under positive selection, and are predicted to impact bacteria-host interactions.