Positive Selection or Free to Vary? Assessing the Functional Significance of Sequence Change Using Molecular Dynamics

Evolutionary arms races between pathogens and their hosts may be manifested as selection for rapid evolutionary change of key genes, and are sometimes detectable through sequence-level analyses. In the case of protein-coding genes, such analyses frequently predict that specific codons are under positive selection. However, detecting positive selection can be non-trivial, and false positive predictions are a common concern in such analyses. It is therefore helpful to place such predictions within a structural and functional context. Here, we focus on the p19 protein from tombusviruses. P19 is a homodimer that sequesters siRNAs, thereby preventing the host RNAi machinery from shutting down viral infection. Sequence analysis of the p19 gene is complicated by the fact that it is constrained at the sequence level by overprinting of a viral movement protein gene. Using homology modeling, in silico mutation and molecular dynamics simulations, we assess how non-synonymous changes to two residues involved in forming the dimer interface—one invariant, and one predicted to be under positive selection—impact molecular function. Interestingly, we find that both observed variation and potential variation (where a non-synonymous change to p19 would be synonymous for the overprinted movement protein) does not significantly impact protein structure or RNA binding. Consequently, while several methods identify residues at the dimer interface as being under positive selection, MD results suggest they are functionally indistinguishable from a site that is free to vary. Our analyses serve as a caveat to using sequence-level analyses in isolation to detect and assess positive selection, and emphasize the importance of also accounting for how non-synonymous changes impact structure and function.     

Canine Evolution in Sabretoothed Carnivores: Natural Selection or Sexual Selection?

The remarkable elongated upper canines of extinct sabretoothed carnivorous mammals have been the subject of considerable speculation on their adaptive function, but the absence of living analogues prevents any direct inference about their evolution. We analysed scaling relationships of the upper canines of 20 sabretoothed feliform carnivores (Nimravidae, Barbourofelidae, Machairodontinae), representing both dirk-toothed and scimitar-toothed sabretooth ecomorphs, and 33 non-sabretoothed felids in relation to body size in order to characterize and identify the evolutionary processes driving their development, using the scaling relationships of carnassial teeth in both groups as a control. Carnassials display isometric allometry in both sabretooths and non-sabretooths, supporting their close relationship with meat-slicing, whereas the upper canines of both groups display positive allometry with body size. Whereas there is no statistical difference in allometry of upper canine height between dirk-toothed and scimitar-toothed sabretooth ecomorphs, the significantly stronger positive allometry of upper canine height shown by sabretooths as a whole compared to non-sabretooths reveals that different processes drove canine evolution in these groups. Although sabretoothed canines must still have been effective for prey capture and processing by hypercarnivorous predators, canine morphology in these extinct carnivores was likely to have been driven to a greater extent by sexual selection than in non-sabretooths. Scaling relationships therefore indicate the probable importance of sexual selection in the evolution of the hypertrophied sabretooth anterior dentition.     

Discord between the Phylogenies Inferred from Molecular versus Functional Data: Uneven Rates of Functional Evolution or Low Levels of Gene Flow?

According to measures of molecular divergence, the three species of the Drosophila simulans clade are closely related to and essentially equidistant from each other. We introgressed 10% of the D. sechellia X chromosome into a pure D. simulans genetic background and found that males carrying this introgressed region were consistently fertile; in contrast, males carrying the same segment from D. mauritiana are sterile and suffer from incompatibilities at a minimum of four loci. Together with other recent results, these data suggest that D. simulans and D. sechellia are much more closely related to each other than either is to D. mauritiana. How can we reconcile the phylogeny inferred from the density of hybrid sterility genes with that inferred from molecular divergence? If the molecular phylogeny is correct, the discrepancy might be explained by uneven rates of functional evolution, resulting in the uneven accumulation of substitutions with corresponding negative effects in hybrids. If the functional phylogeny is correct, then low levels of gene flow across nascent species boundaries, particularly for loci not tightly linked to a hybrid sterility gene, may have erased the original pattern of lineage splitting. We propose tests that will allow us to discriminate between these hypotheses.     

Positive and Negative Selection in the β-Esterase Gene Cluster of the Drosophila melanogaster Subgroup

We examine the pattern of molecular evolution of the β-esterase gene cluster, including the Est-6 and ψEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For ψEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of ψEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of ψEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the β-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function. [Reviewing Editor: Dr. Martin Kreitman]     

Evolution of protein inhibitors of serine proteinases: Positive Darwinian selection or compositional effects?

Summary In at least two instances involving serine proteinase inhibitors it has been shown that functionally important sites evolve faster and exhibit more interspecific variability than functionally neutral sites. Because these phenomena are difficult to reconcile with the neutral theory of molecular evolution, it has been suggested that the accelerated rate of amino acid substitution at the reactive sites is brought about by positive Darwinian selection. We show that differences in the amino acid composition in the different regions of proteinase inhibitors can account for the differences in the rates of amino acid substitution. By using an index of protein mutability [D. Graur (1985) J Mol Evol 2253–62], we show that the amino acid composition of the reactive center in the ovomucoids andSpi-2 gene products is such that, regardless of function, they are expected to evolve more rapidly than any other polypeptide for which the rate of substitution is known. In addition, the reactive region in theSpi-2 proteins is shown to be free of compositional constraint. Positive Darwinian selection need not be invoked at the present time in these cases.     

A Variable Gene in a Conserved Region of the Helicobacter pylori Genome: Isotopic Gene Replacement or Rapid Evolution?

The present study concerns the identification of a novel coding sequence in a region of the Helicobacter pylori genome, located between JHP1069/HP1141 and JHP1071/HP1143 according to the numbering of the J99 and 26695 reference strains, respectively, and spanning three different coding DNA sequences (CDSs). The CDSs located at the centre of this locus were highly polymorphic, as determined by the analysis of 24 European isolates, 3 Asian, and 3 African isolates. Phylogenetic and molecular evolutionary analyses showed that the CDSs were not restricted to the geographical origin of the strains. Despite a very high variability observed in the deduced protein sequences, significant similarity was observed, always with the same protein families, i.e. ATPase and bacteriophage receptor/invasion proteins. Although this variability could be explained by isotopic gene replacement via horizontal transfer of a gene with the same function but coming from a variety of sources, it seems more likely that the very high sequence variation observed at this locus is the result of a strong selection pressure exerted on the corresponding gene product. The CDSs identified in the present study could be used as strain specific markers.     

Evolution of Cryptococcal Antigen Testing: What Is New?

Over the last decade, an upsurge in both the frequency and severity of fungal infections due to the HIV/AIDS epidemic and the use of immunosuppressive therapy has occurred. Even diagnostic methods like culture and microscopy, which have low sensitivity and longer turnaround times, are not widely available, leading to delays in timely antifungal therapy and detrimental patient outcomes. The evolution of cryptococcal antigen (CrAg) testing to develop inexpensive and more sensitive methods to detect cryptococcal antigen is significant. These newer tests employ immunoassays as part of point-of-care platforms, which do not require complex laboratory infrastructure, and they have the potential to detect early disease and reduce time to diagnosis of cryptococcal infection. Advocacy for widely available and efficacious life-saving antifungal treatment should be the only remaining challenge.     

Prevalence of Hepatitis δ (Delta) Virus Infection A Seroepidemiologic Study

In assessing the prevalence of hepatitis δ (delta) virus (HDV) infection in 358 patients with acute hepatitis B seen in Los Angeles between 1983 and 1985 and in 196 patients with chronic hepatitis B followed between 1980 and 1985, we found that 23% of patients with chronic and 5% of patients with acute hepatitis B were infected with HDV. Among patients with chronic hepatitis B, the prevalence of HDV infection was 73% in intravenous drug users and 14% in homosexual men. Acute coinfection with the hepatitis B virus was also more frequent in drug users (8%) than in other groups. δ-Hepatitis is a common infection in hepatitis B virus carriers in Los Angeles, particularly in drug addicts, but also in homosexual men who do not abuse drugs intravenously.     

Nucleotide Sequence Evolution at the κ-Casein Locus: Evidence for Positive Selection within the Family Bovidae

κ-Casein is a mammalian milk protein involved in a number of important physiological processes. In the gut, the ingested protein is split into an insoluble peptide (para κ-casein) and a soluble hydrophilic glycopeptide (caseinomacropeptide). Caseinomacropeptide is responsible for increased efficiency of digestion, prevention of neonate hypersensitivity to ingested proteins, and inhibition of gastric pathogens. Variation within this peptide has significant effects associated with important traits such as milk production. The nucleotide sequences for regions of κ-casein exon and intron four were determined for representatives of the artiodactyl family Bovidae. The pattern of nucleotide substitution in κ-casein sequences for distantly related bovid taxa demonstrates that positive selection has accelerated their divergence at the amino acid sequence level. This selection has differentially influenced the molecular evolution of the two κ-casein split peptides and is focused within a 34-codon region of caseinomacropeptide.     

Detecting Natural Selection at the Molecular Level: A Reexamination of Some “Classic” Examples of Adaptive Evolution

An important criterion used to detect adaptive evolution in DNA sequence data is ω_i > 1, where ω_i is the ratio of nonsynonymous to synonymous substitution rates in lineage i. However, the evaluation of multiple ω_i within a phylogenetic tree can easily inflate the statistical type I error rate. We developed two rigorous methods of analysis that avoid this and other potential pitfalls. We applied these methods to four published examples of adaptive evolution. One case was strongly supported by our reanalysis (abalone sperm lysin), and one was weakly supported (baboon α-globin), but two examples (primate lysozyme and Antarctic fish β-globin) did not show significant evidence of adaptive evolution. Our first method is a “bottom-up” hierarchical maximum likelihood approach, which (1) tests for significant heterogeneity in ω across the phylogeny, (2) locates its source using a sequence of planned comparisons, and (3) tests homogeneous groups of ω for ω > 1, using a modified level of significance that incorporates the pretesting. The second method is a “top-down” log-linear analysis based on estimates of nonsynonymous and synonymous substitutions in pairs of lineages. The log-linear test is applied to pairs of lineages joined at progressively deeper nodes. For each pair, the analysis simultaneously tests for adaptive evolution (ω > 1), a shift in natural selection (ω₁ ≠ω₂), and unequal evolution rate (the relative rate test). In both tests, we emphasized that the criterion ω₁ ≠ ω₂ is an important additional indicator of a phylogenetic shift in the balance between natural selection and genetic drift between two related lineages. [Reviewing Editor: Dr. John Huelsenbeck]     

Can Clues from Evolution Unlock the Molecular Development of the Cerebellum?

The cerebellum sits at the rostral end of the vertebrate hindbrain and is responsible for sensory and motor integration. Owing to its relatively simple architecture, it is one of the most powerful model systems for studying brain evolution and development. Over the last decade, the combination of molecular fate mapping techniques in the mouse and experimental studies, both in vitro and in vivo, in mouse and chick have significantly advanced our understanding of cerebellar neurogenesis in space and time. In amniotes, the most numerous cell type in the cerebellum, and indeed the brain, is the cerebellar granule neurons, and these are born from a transient secondary proliferative zone, the external granule layer (EGL), where proliferation is driven by sonic hedgehog signalling and causes cerebellar foliation. Recent studies in zebrafish and sharks have shown that while the molecular mechanisms of neurogenesis appear conserved across vertebrates, the EGL as a site of shh-driven transit amplification is not, and is therefore implicated as a key amniote innovation that facilitated the evolution of the elaborate foliated cerebella found in birds and mammals. Ellucidating the molecular mechanisms underlying the origin of the EGL in evolution could have significant impacts on our understanding of the molecular details of cerebellar development.     

Evolution of biodiversity: Hyperbola or exponent?

Two models of the evolution of biodiversity during the Phanerozoic are critically analyzed.     

Properties of Antigenomic Hepatitis Delta Virus Ribozyme Cis‐ and Trans‐ Analogs

A series of permuted variants of antigenomic HDV ribozyme and trans‐acting variants were constructed. The catalytic activity study of the ribozymes has shown that all the variants were capable of self‐cleaving with equally biphasic kinetics. Ribonuclease and Fe(II)‐EDTA cleavage have provided evidence that all designed ribozymes fold according to the pseudoknot model and the conformations of the initial and cleaved ribozyme are different. A scheme of HDV ribozyme self‐cleavage reaction was suggested. The role of hydrogen bonds in the reaction was evaluated by substitution of ribose in the ribozyme for deoxyribose. It was found that the 2′‐OH group of U23 and C27 is critical for the reaction to occur; the 2′‐OH group of U32 and U39 is important, while 2′‐OH groups of other nucleotides of loop 3, stem 4 and stem 1 are unimportant for the cleavage activity.