Evolution in vitro: analysis of a lineage of ribozymes

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.     

The ecology and evolution of bacteriocins

In this review we focus on the ecological and evolutionary forces that determine the frequency and diversity of colicins inEscherichia coli. To begin, we describe that this killing phenotype is ubiquitous inE. coli, with as many as 50% of the isolates from a population producing colicin toxins, and that each population sampled has its own unique distribution of the more than 20 known colicin types. Next, we explore the dynamics of colicinogeny, which exhibits a typical form of frequency dependence, where the likelihood of successful colicin invasion into a population increases as the initial density of colicinogenic cells increases. We then incorporate thoughts on the evolution of chromosomal resistance to colicins and describe how resistance might influence the dynamics of colicinogen invasion and maintenance and the resulting colicin diversity. The final section deals with a genetic and phylogenetic characterization of colicins and a discussion of the evolutionary mechanisms responsible for generating colicin diversity. In this final section we provide details of the different molecular mechanisms known to play a role in generating colicin diversity, including the two most dominant forces in colincin evolution: recombination and positive, deversifying, selection.     

learnPopGen: An R package for population genetic simulation and numerical analysis

Here, I briefly present a new R package called learnPopGen that has been designed primarily for the purposes of teaching evolutionary biology, population genetics, and evolutionary theory. Functions of the package can be used to conduct simulations and numerical analyses of a wide range of evolutionary phenomena that would typically be covered in advanced undergraduate through graduate‐level curricula in population genetics or evolution. For instance, learnPopGen functions can be used to visualize gene frequency changes through time under multiple deterministic and stochastic processes, to compute and animate the changes in phenotypic trait values or distributions under natural selection, to numerically analyze and graph the outcome of simple game theory models, and to plot coalescence within a population experiencing genetic drift, along with a number of other things. Functions have been designed to be maximally didactic and frequently employ compelling animated visualizations. Furthermore, it is straightforward to export plots and animations from R in the form of flat or animated graphics, or as videos. For maximum flexibility, students working with the package can run functions directly in R; however, instructors may choose to guide students less adept in the R environment to one of various web interfaces that I have built for a number of the functions of the package and that are already available online.     

Performing Evolution: Role-Play Simulations

By simulating evolution through performance, students become physically, as well as mentally, engaged in thinking about evolutionary concepts. This instructional strategy redirects tension around the subject toward metacognitive reflection. Non-verbal performances like those presented here also avoid the pitfalls of relying on difficult-to-use language. This paper describes a teachable unit including the learning goals and outcomes as well as rubrics to aid assessment. Through two performance-based activities, the unit introduces the fundamental evolutionary concepts that evolution lacks forethought and that natural selection is a sorting process. By reflecting on the performances, students learn other sophisticated evolutionary concepts like hitchhiking, the effects of environmental change, and the extinction of traits. They also become aware of the scientific process, articulating hypotheses about the outcome of the simulations, collecting data, and revising their hypotheses. Discussions and homework about the performances reveal how learning progresses, and detailed rubrics help both instructors and students assess conceptual learning. This unit concludes with the opportunity for students to transfer what they have learned to new concepts: they design new performances to simulate other mechanisms of evolution, such as genetic drift, mutation, and migration.     

Allelic heterogeneity and trade-off shape natural variation for response to soil micronutrient

As sessile organisms, plants have to cope with diverse environmental constraints that may vary through time and space, eventually leading to changes in the phenotype of populations through fixation of adaptive genetic variation. To fully comprehend the mechanisms of evolution and make sense of the extensive genotypic diversity currently revealed by new sequencing technologies, we are challenged with identifying the molecular basis of such adaptive variation. Here, we have identified a new variant of a molybdenum (Mo) transporter, MOT1, which is causal for fitness changes under artificial conditions of both Mo-deficiency and Mo-toxicity and in which allelic variation among West-Asian populations is strictly correlated with the concentration of available Mo in native soils. In addition, this association is accompanied at different scales with patterns of polymorphisms that are not consistent with neutral evolution and show signs of diversifying selection. Resolving such a case of allelic heterogeneity helps explain species-wide phenotypic variation for Mo homeostasis and potentially reveals trade-off effects, a finding still rarely linked to fitness.     

The evolution of low mutation rates in experimental mutator populations of Saccharomyces cerevisiae

Mutation is the source of both beneficial adaptive variation and deleterious genetic load, fueling the opposing selective forces than shape mutation rate evolution. This dichotomy is well illustrated by the evolution of the mutator phenotype, a genome-wide 10- to 100-fold increase in mutation rate. This phenotype has often been observed in clonally expanding populations exposed to novel or frequently changing conditions. Although studies of both experimental and natural populations have shed light on the evolutionary forces that lead to the spread of the mutator allele through a population, significant gaps in our understanding of mutator evolution remain. Here we use an experimental evolution approach to investigate the conditions required for the evolution of a reduction in mutation rate and the mechanisms by which populations tolerate the accumulation of deleterious mutations. We find that after ～6,700 generations, four out of eight experimental mutator lines had evolved a decreased mutation rate. We provide evidence that the accumulation of deleterious mutations leads to selection for reduced mutation rate clones in populations of mutators. Finally, we test the long-term consequences of the mutator phenotype, finding that mutator lines follow different evolutionary trajectories, some of which lead to drug resistance.     

Using online lectures to make time for active learning

To make time in class for group activities devoted to critical thinking, we integrated a series of short online lectures into the homework assignments of a large, introductory biology course at a research university. The majority of students viewed the online lectures before coming to class and reported that the online lectures helped them to complete the in-class activity and did not increase the amount of time they devoted to the course. In addition, students who viewed the online lecture performed better on clicker questions designed to test lower-order cognitive skills. The in-class activities then gave the students practice analyzing the information in groups and provided the instructor with feedback about the students' understanding of the material. On the basis of the results of this study, we support creating hybrid course models that allow students to learn the fundamental information outside of class time, thereby creating time during the class period to be dedicated toward the conceptual understanding of the material.     

A rheostat model for a rapid and reversible form of imprinting-dependent evolution

The evolutionary advantages of genomic imprinting are puzzling. We propose that genomic imprinting evolved as a mechanism that maximizes the interindividual variability in the rates of gene expression for dosage-sensitive loci that, with minimal unrelated deleterious effects, can alter the phenotype over a wide continuum. We hypothesize (1) that genomic imprinting provides a previously suggested haploid selective advantage (HSA); (2) that many imprinted genes have evolved mechanisms that facilitate quantitative hypervariability (QH) of gene expression; (3) that the combination of HSA and QH makes possible a rapid and reversible form of imprinting-dependent evolution (IDE) that can mediate changes in phenotype; and (4) that this enhanced adaptability to a changing environment provides selective advantage to the population, as an assisted form of evolution. These mechanisms may have provided at least one of the driving forces for the evolution of genomic imprinting in mammals. The rheostat model suggests that both genetic and epigenetic variants can contribute to an integrated mechanism of mixed Mendelian and non-Mendelian inheritance and suggests the possibility that the majority of variants are not intrinsically deleterious but, depending on the environment, are each potentially advantageous. Moreover, this would be a reversible form of evolution, with the ability not only to protect a silent allele from selection for many generations but to reactivate and expand it in the population quickly.     

Novel gain-of-function alleles demonstrate a role for the heterochronic gene lin-41 in C. elegans male tail tip morphogenesis

To gain an understanding of the genes and mechanisms that govern morphogenesis and its evolution, we have analyzed mutations that disrupt this process in a simple model structure, the male tail tip of the rhabditid nematode C. elegans. During the evolution of rhabditid male tails, there have been several independent changes from tails with rounded tips ("peloderan", as in C. elegans) to those with pointed tips ("leptoderan"). Mutations which produce leptoderan (Lep) tails in C. elegans thus identify candidate genes and pathways in which evolutionary changes could have produced leptoderan tails from peloderan ancestors. Here we report that two novel, gain-of-function (gf) alleles of lin-41 have lesions predicted to affect the N-terminus of the RBCC-domain LIN-41 protein. Both gf alleles cause the tail tip of adult males to retain the pointed shape of the juvenile tails, producing a Lep phenotype that looks like the tails of leptoderan species. Consistent with its role in the heterochronic pathway, we find that lin-41 governs the timing and extent of male tail tip morphogenesis in a dose-dependent manner. Specifically, the Lep phenotype results from a heterochronic delay in the retraction and fusion of the tail tip cells during L4 morphogenesis, such that retraction is not completed before the adult molt. Conversely, we find that tail tip morphogenesis and cell fusions begin precociously at the L3 stage in the reduced-function lin-41 mutant, ma104, resulting in over-retracted male tails in the adult. Because modulated anti-LIN-41 RNAi knockdowns in the gf mutants restore wild-type phenotype, we suggest that the leptoderan phenotype of the gf alleles is due to a higher activity of otherwise normal LIN-41. Additionally, the gf allele is suppressed by the wild-type allele, suggesting that LIN-41 normally regulates itself, possibly by autoubiquitination. We speculate that small changes affecting LIN-41 could have been significant for male tail evolution.     

A Genetic Strategy to Demonstrate the Occurrence of Spontaneous Mutations in Nondividing Cells within Colonies of Escherichia Coli

A genetic strategy was designed to examine the occurrence of mutations in stationary-phase populations. In this strategy, a parental population of cells is able to survive under both permissive and restrictive conditions whereas mutants at a particular target locus exhibit a conditional-lethal phenotype. Thus, by growing the population to stationary phase under restrictive conditions and then shifting it to permissive conditions, mutations that had arisen in stationary phase can be studied without confounding effects caused by the occurrence of similar mutations during growth of the population. In two different applications of this strategy, we have studied the reversion to Lac(+) in stationary phase of several Lac(-) mutations in Escherichia coli. Our results indicate that a variety of spontaneous point mutations and deletions, particularly those that are sensitive to the mechanisms of replication slippage (for their generation) and methyl-directed mismatch repair (for their correction), can arise in nondividing populations of cells within a colony. The frequency of their occurrence was also elevated in mutS strains, which are defective in such mismatch repair. These data have relevance to the ongoing debate on adaptive or directed mutations in bacteria.     

Quantification and decomposition of environment‐selection relationships

In nature, selection varies across time in most environments, but we lack an understanding of how specific ecological changes drive this variation. Ecological factors can alter phenotypic selection coefficients through changes in trait distributions or individual mean fitness, even when the trait‐absolute fitness relationship remains constant. We apply and extend a regression‐based approach in a population of Soay sheep (Ovis aries) and suggest metrics of environment‐selection relationships that can be compared across studies. We then introduce a novel method that constructs an environmentally structured fitness function. This allows calculation of full (as in existing approaches) and partial (acting separately through the absolute fitness function slope, mean fitness, and phenotype distribution) sensitivities of selection to an ecological variable. Both approaches show positive overall effects of density on viability selection of lamb mass. However, the second approach demonstrates that this relationship is largely driven by effects of density on mean fitness, rather than on the trait‐fitness relationship slope. If such mechanisms of environmental dependence of selection are common, this could have important implications regarding the frequency of fluctuating selection, and how previous selection inferences relate to longer term evolutionary dynamics.     

Dosage balance in gene regulation: biological implications

Classical studies in genetics involving aneuploidy and ploidy comparisons and sex-determination mechanisms indicated a balance phenomenon such that changes of individual chromosomal dosage altered the phenotype more dramatically than changes in ploidy. Recent evidence suggests that a major contributor to this balance is the behavior of molecular complexes that function in various regulatory processes affecting gene expression. In this article, we discuss the potential contribution of regulatory balance to the control of quantitative traits, hybrid vigor, genome evolution and post-zygotic speciation mechanisms.     

Evolution of dominance in metabolic pathways

Dominance is a form of phenotypic robustness to mutations. Understanding how such robustness can evolve provides a window into how the relation between genotype and phenotype can evolve. As such, the issue of dominance evolution is a question about the evolution of inheritance systems. Attempts at explaining the evolution of dominance have run into two problems. One is that selection for dominance is sensitive to the frequency of heterozygotes. Accordingly, dominance cannot evolve unless special conditions lead to the presence of a high frequency of mutant alleles in the population. Second, on the basis of theoretical results in metabolic control analysis, it has been proposed that metabolic systems possess inherent constraints. These hypothetical constraints imply the default manifestation of dominance of the wild type with respect to the effects of mutations at most loci. Hence, some biologists have maintained that an evolutionary explanation is not relevant to dominance. In this article, we put into question the hypothetical assumption of default metabolic constraints. We show that this assumption is based on an exclusion of important nonlinear interactions that can occur between enzymes in a pathway. With an a priori exclusion of such interactions, the possibility of epistasis and hence dominance modification is eliminated. We present a theoretical model that integrates enzyme kinetics and population genetics to address dominance evolution in metabolic pathways. In the case of mutations that decrease enzyme concentrations, and given the mechanistic constraints of Michaelis-Menten-type catalysis, it is shown that dominance of the wild type can be extensively modified in a two-enzyme pathway. Moreover, we discuss analytical results indicating that the conclusions from the two-enzyme case can be generalized to any number of enzymes. Dominance modification is achieved chiefly through changes in enzyme concentrations or kinetic parameters such as k(cat), both of which can alter saturation levels. Low saturation translates into higher levels of dominance with respect to mutations that decrease enzyme concentrations. Furthermore, it is shown that in the two-enzyme example, dominance evolves as a by-product of selection in a manner that is insensitive to the frequency of heterozygotes. Using variation in k(cat) as an example of modifier mutations, it is shown that the latter can have direct fitness effects in addition to dominance modification effects. Dominance evolution can occur in a frequency-insensitive manner as a result of selection for such dual-effects alleles. This type of selection may prove to be a common pattern for the evolution of phenotypic robustness to mutations.     

The evolution of endothermy: role for membranes and molecular activity

On the basis of the comparative approach and three models of metabolism (endothermic and ectothermic vertebrates, body mass, and mammalian development), we suggest that a few common cellular processes, linked either directly or indirectly to membranes, consume the majority of energy used by most organisms; that membranes act as pacemakers of metabolism through changes in lipid composition, altering membrane characteristics and the working environment of membrane proteins--specifically, that changes in the membrane environment similarly affect the molecular activities (specific rates of activity) of membrane-bound proteins; and that polyunsaturation of membranes increases whereas monounsaturation decreases the activity of membrane proteins. Experiments designed to test this theory using the sodium pump support this supposition. Potential mechanisms considered include fluidity, electrical fields, and related surface area requirements of lipids. In considering the evolution of endothermy in mammals, for example, if the first mammals were small, possibly nocturnal and active organisms, all these factors would favour increased polyunsaturation of membranes. Such changes (from monounsaturated to polyunsaturated membranes) would allow membranes to set the pace of metabolism in the evolution of endothermy.     

The ultimate and proximate mechanisms driving the evolution of long tails in forest deer mice

Understanding both the role of selection in driving phenotypic change and its underlying genetic basis remain major challenges in evolutionary biology. Here, we use modern tools to revisit a classic system of local adaptation in the North American deer mouse, Peromyscus maniculatus, which occupies two main habitat types: prairie and forest. Using historical collections, we find that forest‐dwelling mice have longer tails than those from nonforested habitat, even when we account for individual and population relatedness. Using genome‐wide SNP data, we show that mice from forested habitats in the eastern and western parts of their range form separate clades, suggesting that increased tail length evolved independently. We find that forest mice in the east and west have both more and longer caudal vertebrae, but not trunk vertebrae, than nearby prairie forms. By intercrossing prairie and forest mice, we show that the number and length of caudal vertebrae are not correlated in this recombinant population, indicating that variation in these traits is controlled by separate genetic loci. Together, these results demonstrate convergent evolution of the long‐tailed forest phenotype through two distinct genetic mechanisms, affecting number and length of vertebrae, and suggest that these morphological changes—either independently or together—are adaptive.     

The temporal distribution of directional gradients under selection for an optimum

Temporal variation in phenotypic selection is often attributed to environmental change causing movements of the adaptive surface relating traits to fitness, but this connection is rarely established empirically. Fluctuating phenotypic selection can be measured by the variance and autocorrelation of directional selection gradients through time. However, the dynamics of these gradients depend not only on environmental changes altering the fitness surface, but also on evolution of the phenotypic distribution. Therefore, it is unclear to what extent variability in selection gradients can inform us about the underlying drivers of their fluctuations. To investigate this question, we derive the temporal distribution of directional gradients under selection for a phenotypic optimum that is either constant or fluctuates randomly in various ways in a finite population. Our analytical results, combined with population‐ and individual‐based simulations, show that although some characteristic patterns can be distinguished, very different types of change in the optimum (including a constant optimum) can generate similar temporal distributions of selection gradients, making it difficult to infer the processes underlying apparent fluctuating selection. Analyzing changes in phenotype distributions together with changes in selection gradients should prove more useful for inferring the mechanisms underlying estimated fluctuating selection.     

Expanding course goals beyond disciplinary boundaries: physiology education in an undergraduate course on psychoactive drugs

The topic of psychoactive drugs is one of inherent interest to college students. We used this insight to design and implement a multidisciplinary undergraduate course with psychoactive drugs as the central theme. The Medical Science of Psychoactive Drugs examines the biological mechanisms underlying all major effects of psychoactive drugs, including the effects on the brain and other organs and tissues. Physiological principles, molecular mechanisms, and genetic factors involved in drug-induced therapeutic and adverse effects are emphasized. The course is open to undergraduate students at all levels and carries no prerequisites, and enrollment is limited to approximately 50 students. Major teaching modes include lecture, short homework papers on topics related to the previous class meeting, small-group discussions at several points during each class, and whole class discussions. Because of the diversity of students' knowledge of basic science, we employ a variety of methods designed to help students grasp the necessary scientific concepts. Our methods are intended to be inquiry based and highly interactive. Our goals are 1) to foster the development of an organized knowledge base about psychoactive drugs that will have practical applicability in the daily lives of the students; 2) to promote the rational application of this knowledge in thinking about current medical, social, legal, and ethical issues involving psychoactive drugs; and 3) to cultivate science literacy, critical thinking, and communication skills among students.     

Genomic instability in picornaviruses

Picornaviruses are small animal RNA viruses and include wtiological agents of poliomyelitis, foot and mouse disease, hepatitis A, etc. Replication of their genome results in many mutations, which are close in number to a viability threshold. Hence every virus population contains a great variety of genomes and represents a quasispecies. Covalent rearrangements (deletions, insertions, recombination) also contribute to genome variation and arise by replicative and nonreplicative mechanisms, which are still poorly understood. Only a minor fraction of all new changes is fixed during evolution. The fixation is based on two principally different ways of selection: with (positive and negative selection) and without (random selection of nonrepresentative variants) regard to the phenotype. In natural evolution of picornaviruses, the latter way is prevalent, and most fixed mutations are phenotypically neutral. To understand the mechanisms of evolution, it is necessary to evaluate the biological significance of particular genetic changes. Several new approaches to this problem have recently been proposed.