Adaptive protein evolution in Drosophila

For over 30 years a central question in molecular evolution has been whether natural selection plays a substantial role in evolution at the DNA sequence level. Evidence has accumulated over the last decade that adaptive evolution does occur at the protein level, but it has remained unclear how prevalent adaptive evolution is. Here we present a simple method by which the number of adaptive substitutions can be estimated and apply it to data from Drosophila simulans and D. yakuba. We estimate that 45% of all amino-acid substitutions have been fixed by natural selection, and that on average one adaptive substitution occurs every 45 years in these species.     

The cost of inbreeding in Arabidopsis

Population geneticists have long sought to estimate the distribution of selection intensities among genes of diverse function across the genome. Only recently have DNA sequencing and analytical techniques converged to make this possible. Important advances have come from comparing genetic variation within species (polymorphism) with fixed differences between species (divergence). These approaches have been used to examine individual genes for evidence of selection. Here we use the fact that the time since species divergence allows combination of data across genes. In a comparison of amino-acid replacements among species of the mustard weed Arabidopsis with those among species of the fruitfly Drosophila, we find evidence for predominantly beneficial gene substitutions in Drosophila but predominantly detrimental substitutions in Arabidopsis. We attribute this difference to the Arabidopsis mating system of partial self-fertilization, which corroborates a prediction of population genetics theory that species with a high frequency of inbreeding are less efficient in eliminating deleterious mutations owing to their reduced effective population size.     

The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration.

Three dominant mutations of mec-4, a gene needed for mechanosensation, cause the touch-receptor neurons of Caenorhabditis elegans to degenerate. With deg-1, another C. elegans gene that can mutate to induce neuronal degeneration and that is similar in sequence, mec-4 defines a new gene family. Cross-hybridizing sequences are detectable in other species, raising the possibility that degenerative conditions in other organisms may be caused by mutations in similar genes. All three dominant mec-4 mutations affect the same amino acid. Effects of amino-acid substitutions at this position suggest that steric hindrance may induce the degenerative state.     

Positive selection at sites of multiple amino acid replacements since rat-mouse divergence

New alleles become fixed owing to random drift of nearly neutral mutations or to positive selection of substantially advantageous mutations. After decades of debate, the fraction of fixations driven by selection remains uncertain. Within 9,390 genes, we analysed 28,196 codons at which rat and mouse differ from each other at two nucleotide sites and 1,982 codons with three differences. At codons where rat-mouse divergence involved two non-synonymous substitutions, both of them occurred in the same lineage, either rat or mouse, in 64% of cases; however, independent substitutions would occur in the same lineage with a probability of only 50%. All three non-synonymous substitutions occurred in the same lineage for 46% of codons, instead of the 25% expected. Furthermore, comparison of 12 pairs of prokaryotic genomes also shows clumping of multiple non-synonymous substitutions in the same lineage. This pattern cannot be explained by correlated mutation or episodes of relaxed negative selection, but instead indicates that positive selection acts at many sites of rapid, successive amino acid replacement.     

Testing the neutral theory of molecular evolution with genomic data from Drosophila

Although positive selection has been detected in many genes, its overall contribution to protein evolution is debatable. If the bulk of molecular evolution is neutral, then the ratio of amino-acid (A) to synonymous (S) polymorphism should, on average, equal that of divergence. A comparison of the A/S ratio of polymorphism in Drosophila melanogaster with that of divergence from Drosophila simulans shows that the A/S ratio of divergence is twice as high---a difference that is often attributed to positive selection. But an increase in selective constraint owing to an increase in effective population size could also explain this observation, and, if so, all genes should be affected similarly. Here we show that the difference between polymorphism and divergence is limited to only a fraction of the genes, which are also evolving more rapidly, and this implies that positive selection is responsible. A higher A/S ratio of divergence than of polymorphism is also observed in other species, which suggests a rate of adaptive evolution that is far higher than permitted by the neutral theory of molecular evolution.     

同义密码子的频率分布

本文提出了关于同义密码子非无规分布的一个机制,从主方程出发,如果只考虑同义突变由图论方法证明了密码子的均匀分布,但是如果考虑了不同氮基酸间的突变,便可导出同义密码子的非无规分布。后者反映了选择约束对密码子分布的影响。     

分子进化中性理论的进展

中性理论与达尔文的自然选择进化论有明显的差别。中性论认为在分子水平上覆盖着多数的进化改变，是在连续产生突变时，由于选择中性（即选择相等），使突变得到随机的固定（即小群体内，样本的随机漂变）。还认为在分子水平上，大多数的种内变异（如蛋白质和ＤＮＡ多态）是选择中性或近于中性，它们能够保持在种内是由于突变的产生和随机消失之间的平衡引起的。近年来，ＤＮＡ顺序的研究资料，都有力地支持中性论，如鼠的拟球蛋白基因、鼹鼠的αＡ－晶状体蛋白、流行性感冒Ａ病毒基因，果蝇的核和线粒体基因等。所以，从地球上生命的历史来看，中性进化的改变至少在数量上已超过达尔文自然选择进化的改变。     

Interference among deleterious mutations favours sex and recombination in finite populations

Sex and recombination are widespread, but explaining these phenomena has been one of the most difficult problems in evolutionary biology. Recombination is advantageous when different individuals in a population carry different advantageous alleles. By bringing together advantageous alleles onto the same chromosome, recombination speeds up the process of adaptation and opposes the fixation of harmful mutations by means of Muller's ratchet. Nevertheless, adaptive substitutions favour sex and recombination only if the rate of adaptive mutation is high, and Muller's ratchet operates only in small or asexual populations. Here, by tracking the fate of modifier alleles that alter the frequency of sex and recombination, we show that background selection against deleterious mutant alleles provides a stochastic advantage to sex and recombination that increases with population size. The advantage arises because, with low levels of recombination, selection at other loci severely reduces the effective population size and genetic variance in fitness at a focal locus (the Hill-Robertson effect), making a population less able to respond to selection and to rid itself of deleterious mutations. Sex and recombination reveal the hidden genetic variance in fitness by combining chromosomes of intermediate fitness to create chromosomes that are relatively free of (or are loaded with) deleterious mutations. This increase in genetic variance within finite populations improves the response to selection and generates a substantial advantage to sex and recombination that is fairly insensitive to the form of epistatic interactions between deleterious alleles. The mechanism supported by our results offers a robust and broadly applicable explanation for the evolutionary advantage of recombination and can explain the spread of costly sex.     

Evolutionary genomics: detecting selection needs comparative data

Positive selection at the molecular level is usually indicated by an increase in the ratio of non-synonymous to synonymous substitutions (dN/dS) in comparative data. However, Plotkin et al. describe a new method for detecting positive selection based on a single nucleotide sequence. We show here that this method is particularly sensitive to assumptions regarding the underlying mutational processes and does not provide a reliable way to identify positive selection.     

Adaptive evolution of non-coding DNA in Drosophila

A large fraction of eukaryotic genomes consists of DNA that is not translated into protein sequence, and little is known about its functional significance. Here I show that several classes of non-coding DNA in Drosophila are evolving considerably slower than synonymous sites, and yet show an excess of between-species divergence relative to polymorphism when compared with synonymous sites. The former is a hallmark of selective constraint, but the latter is a signature of adaptive evolution, resembling general patterns of protein evolution in Drosophila. I estimate that about 40-70% of nucleotides in intergenic regions, untranslated portions of mature mRNAs (UTRs) and most intronic DNA are evolutionarily constrained relative to synonymous sites. However, I also use an extension to the McDonald-Kreitman test to show that a substantial fraction of the nucleotide divergence in these regions was driven to fixation by positive selection (about 20% for most intronic and intergenic DNA, and 60% for UTRs). On the basis of these observations, I suggest that a large fraction of the non-translated genome is functionally important and subject to both purifying selection and adaptive evolution. These results imply that, although positive selection is clearly an important facet of protein evolution, adaptive changes to non-coding DNA might have been considerably more common in the evolution of D. melanogaster.     

Allele substitution at a flower colour locus produces a pollinator shift in monkeyflowers

The role of major mutations in adaptive evolution has been debated for more than a century. The classical view is that adaptive mutations are nearly infinite in number with infinitesimally small phenotypic effect, but recent theory suggests otherwise. To provide empirical estimates of the magnitude of adaptive mutations in wild plants, we conducted field studies to determine the adaptive value of alternative alleles at a single locus, YELLOW UPPER (YUP). YUP controls the presence or absence of yellow carotenoid pigments in the petals of pink-flowered Mimulus lewisii, which is pollinated by bumblebees, and its red-flowered sister species M. cardinalis, which is pollinated by hummingbirds. We bred near-isogenic lines (NILs) in which the YUP allele from each species was substituted into the other. M. cardinalis NILs with the M. lewisii YUP allele had dark pink flowers and received 74-fold more bee visits than the wild type, whereas M. lewisii NILs with the M. cardinalis yup allele had yellow-orange flowers and received 68-fold more hummingbird visits than the wild type. These results indicate that an adaptive shift in pollinator preference may be initiated by a single major mutation.     

Can the product of the theta gene be a real globin?

A new member (theta 1, or psi alpha) of the alpha-globin gene family has recently been identified in a number of species. In higher primates the theta 1 gene has all the structural features apparently necessary for expression, and it appears to have long been under strong selective constraints which suggests that it could still be, or recently have been, a functional gene. No corresponding 'globin' has yet been identified, however. In some other species, galago and rabbit for example, the theta 1 and psi alpha genes have accumulated enough inactivating mutations for them to be considered genuine pseudogenes. Horses also have an alpha-like gene (psi alpha), in a 3' position identical to the other species in relation to the functional alpha genes, and this also appears to have the elements required for a functional gene. The predicted amino-acid sequence, however, suggests that any 'globin' product is likely to be non-viable because it has a number of seriously deleterious amino-acid replacements. Some of these amino-acid changes are shared with the rabbit and primate sequences, indicating that they predate the mammalian radiation, and that if indeed any of these genes are still functional, they are unlikely to be making haemoglobin.     

Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein

The marginal net stability of a folded protein is thought to depend on a small difference between large, compensating individual forces. Therefore, the net free energy of stabilization of proteins is unexpectedly small (approximately 10 kcal mol-1). The contribution of individual forces such as hydrogen bonds and salt bridges to the stabilization is evaluated as 1-3 kcal mol-1, and several additional forces are thought to be sufficient to account for the extra thermostability of thermophilic proteins. The native conformation of a protein is determined by the total number of interatomic interactions and hence by the amino-acid sequence. If the few amino-acid residues that individually contribute to the stabilization could be implemented concurrently into the sequence, the multiple replacement would enhance the overall stability of the protein molecule. Here we report evidence to support this argument. Thermal inactivation kinetics and proteolytic resistance for mutants of a kanamycin nucleotidyltransferase reveal that a few intragenic amino-acid replacements stabilize the protein cumulatively. Our experiments not only demonstrate the feasibility of elevating the thermostability of a protein but also lead to better understanding of the forces that are responsible for protein stability.     

Alteration in crossbridge kinetics caused by mutations in actin

The generation of force during muscle contraction results from the interaction of myosin and actin. The kinetics of this force generation vary between different muscle types and within the same muscle type in different species. Most attention has focused on the role of myosin isoforms in determining these differences. The role of actin isoforms has received little attention, largely because of the lack of a suitable cell type in which the myosin isoform remains constant yet the actin isoforms vary. An alternative approach would be to examine the effect of actin mutations, however, most of these cause such gross disruption of muscle structure that mechanical measurements are impossible. We have now identified two actin mutations which, despite involving conserved amino acids, can assemble into virtually normal myofibrils. These amino-acid changes in actin significantly affect the kinetics of force generation by muscle fibres. One of the mutations is not in the putative myosin-binding site, demonstrating the importance of long-range effects of amino acids on actin function.     

Positive selection of a gene family during the emergence of humans and African apes

Gene duplication followed by adaptive evolution is one of the primary forces for the emergence of new gene function. Here we describe the recent proliferation, transposition and selection of a 20-kilobase (kb) duplicated segment throughout 15 Mb of the short arm of human chromosome 16. The dispersal of this segment was accompanied by considerable variation in chromosomal-map location and copy number among hominoid species. In humans, we identified a gene family (morpheus) within the duplicated segment. Comparison of putative protein-encoding exons revealed the most extreme case of positive selection among hominoids. The major episode of enhanced amino-acid replacement occurred after the separation of human and great-ape lineages from the orangutan. Positive selection continued to alter amino-acid composition after the divergence of human and chimpanzee lineages. The rapidity and bias for amino-acid-altering nucleotide changes suggest adaptive evolution of the morpheus gene family during the emergence of humans and African apes. Moreover, some genes emerge and evolve very rapidly, generating copies that bear little similarity to their ancestral precursors. Consequently, a small fraction of human genes may not possess discernible orthologues within the genomes of model organisms.     

A new way of enhancing the thermostability of proteases

Thermostability of enzyme can be enhanced by single amino acid substitutions. Recent advances in genetic engineering have made it possible to create novel proteins in a predictable manner where structural information for the protein is available. This 'protein engineering' has already been used to enhance enzyme thermostability, but it is usually not clear which amino acid substitutions should be made. We consider that the following approach should be helpful in engineering proteins with enhanced thermostability: highly conserved residues should be left unchanged; the sequences of known mesophilic and thermophilic proteins should be used to suggest the kinds of changes likely to increase thermostability; and substitutions should be made that increase internal hydrophobicity and that stabilize helices for strong internal packing. We describe here the use of this approach to alter the thermostability of the thermostable neutral protease of Bacillus stearothermophilus, the sequence of which is known. Surprisingly we find that a single mutation that decreases thermostability can require two mutations that increase stability to compensate for it. The effects on stability are not additive, suggesting cooperativity.     

Calcium channel characteristics conferred on the sodium channel by single mutations.

The sodium channel, one of the family of structurally homologous voltage-gated ion channels, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which occurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in determining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.     

Reduced adaptation of a non-recombining neo-Y chromosome

Sex chromosomes are generally believed to have descended from a pair of homologous autosomes. Suppression of recombination between the ancestral sex chromosomes led to the genetic degeneration of the Y chromosome. In response, the X chromosome may become dosage-compensated. Most proposed mechanisms for the degeneration of Y chromosomes involve the rapid fixation of deleterious mutations on the Y. Alternatively, Y-chromosome degeneration might be a response to a slower rate of adaptive evolution, caused by its lack of recombination. Here we report patterns of DNA polymorphism and divergence at four genes located on the neo-sex chromosomes of Drosophila miranda. We show that a higher rate of protein sequence evolution of the neo-X-linked copy of Cyclin B relative to the neo-Y copy is driven by positive selection, which is consistent with the adaptive hypothesis for the evolution of the Y chromosome. In contrast, the neo-Y-linked copies of even-skipped and roundabout show an elevated rate of protein evolution relative to their neo-X homologues, probably reflecting the reduced effectiveness of selection against deleterious mutations in a non-recombining genome. Our results provide evidence for the importance of sexual recombination for increasing and maintaining the level of adaptation of a population.