Characterization of T4 Mutants That Partially Suppress the Inability of T4rII to Grow in Lambda Lysogens

In the course of isolating viable T4 deletions that affect plaque morphology (Homyk and Weil 1974), two closely linked point mutants, sip1 and sip2, were obtained. They map between genes t and 52, cause a reduction in plaque size and burst size, and partially suppress the lethality of rII mutants for growth in lambda lysogens. These characteristics demonstrate that sip1 and sip2 are similar to mutants previously reported by Freedman and Brenner (1972). In addition, D. Hall (personal communication) has shown that sip1 and sip2 are similar to the mutant farP85, which affects the regulation of a number of early genes ( Chace and Hall 1975).——Sip suppression of rII mutants can be demonstrated in one-step growth experiments, even when both rII genes are completely deleted. This indicates that sip mutants do not simply reduce the level of rII gene products required for growth in a lambda lysogen. Instead, they alter the growth cycle so as to partially circumvent the need for any rII products.——Mutations at two other sites, designated L₁ and L₂, reverse the poor phage growth caused by sip and, in the one case tested, reverse the rII-suppressing ability of sip.     

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K _m = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (K _m = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.     

Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes

L-arginine (L-Arg) deficiency results in decreased T-cell proliferation and impaired T-cell function. Here we have found that L-Arg depletion inhibited expression of different membrane antigens, including CD247 (CD3ζ), and led to an ER stress response, as well as cell cycle arrest at G₀/G₁ in both human Jurkat and peripheral blood mitogen-activated T cells, without undergoing apoptosis. By genetic and biochemical approaches, we found that L-Arg depletion also induced autophagy. Deprivation of L-Arg induced EIF2S1 (eIF2α), MAPK8 (JNK), BCL2 (Bcl-2) phosphorylation, and displacement of BECN1 (Beclin 1) binding to BCL2, leading to autophagosome formation. Silencing of ERN1 (IRE1α) prevented the induction of autophagy as well as MAPK8 activation, BCL2 phosphorylation and XBP1 splicing, whereas led T lymphocytes to apoptosis under L-Arg starvation, suggesting that the ERN1-MAPK8 pathway plays a major role in the activation of autophagy following L-Arg depletion. Autophagy was required for survival of T lymphocytes in the absence of L-Arg, and resulted in a reversible process. Replenishment of L-Arg made T lymphocytes to regain the normal cell cycle profile and proliferate, whereas autophagy was inhibited. Inhibition of autophagy by ERN1, BECN1 and ATG7 silencing, or by pharmacological inhibitors, promoted cell death of T lymphocytes incubated in the absence of L-Arg. Our data indicate for the first time that depletion of L-Arg in T lymphocytes leads to a reversible response that preserves T lymphocytes through ER stress and autophagy, while remaining arrested at G₀/G₁. Our data also show that the L-Arg depletion-induced ER stress response could lead to apoptosis when autophagy is blocked.     

Estimating the Parameters of Selection on Nonsynonymous Mutations in Drosophila pseudoobscura and D. miranda

We present the results of surveys of diversity in sets of >40 X-linked and autosomal loci in samples from natural populations of Drosophila miranda and D. pseudoobscura, together with their sequence divergence from D. affinis. Mean silent site diversity in D. miranda is approximately one-quarter of that in D. pseudoobscura; mean X-linked silent diversity is about three-quarters of that for the autosomes in both species. Estimates of the distribution of selection coefficients against heterozygous, deleterious nonsynonymous mutations from two different methods suggest a wide distribution, with coefficients of variation greater than one, and with the average segregating amino acid mutation being subject to only very weak selection. Only a small fraction of new amino acid mutations behave as effectively neutral, however. A large fraction of amino acid differences between D. pseudoobscura and D. affinis appear to have been fixed by positive natural selection, using three different methods of estimation; estimates between D. miranda and D. affinis are more equivocal. Sources of bias in the estimates, especially those arising from selection on synonymous mutations and from the choice of genes, are discussed and corrections for these applied. Overall, the results show that both purifying selection and positive selection on nonsynonymous mutations are pervasive.SURVEYS of DNA sequence diversity and divergence are shedding light on a number of questions in evolutionary genetics (for recent reviews, see Akey 2009; Sella et al. 2009). Two of the most important questions of this kind concern the distribution of selection coefficients against deleterious mutations affecting protein sequences and the proportion of amino acid sequence differences between related species that have been fixed by positive selection. Several different methods have been proposed for studying each of these questions, using different features of data on polymorphism and divergence at nonsynonymous and silent sites.For example, the parameters of the distribution of selection coefficients against deleterious amino acid mutations have been estimated by contrasting the numbers of nonsynonymous and silent within-species polymorphisms and fixed differences between species (Sawyer and Hartl 1992; Bustamante et al. 2002; Piganeau and Eyre-Walker 2003; Sawyer et al. 2007); by fitting the frequency spectra of nonsynonymous and silent variants to models of selection, mutation, and drift (Akashi 1999; Eyre-Walker et al. 2006; Keightley and Eyre-Walker 2007; Kryukov et al. 2007; Boyko et al. 2008; Eyre-Walker and Keightley 2009); or by comparing levels of nonsynonymous and silent diversities between species with different population sizes (Loewe and Charlesworth 2006; Loewe et al. 2006). The results of these different approaches generally agree in suggesting that there is a wide distribution of selection coefficients against nonsynonymous mutations and that the mean selection coefficient against heterozygous carriers of such mutations is very small. The results imply that a typical individual from a human population carries several hundred weakly deleterious mutations (Eyre-Walker et al. 2006; Kryukov et al. 2007; Boyko et al. 2008); for a typical Drosophila population, with its much higher level of variability, the number is probably an order of magnitude greater (Loewe et al. 2006; Keightley and Eyre-Walker 2007).The presence of this large load of slightly deleterious mutations in human and natural populations, most of which are held at low frequencies by natural selection, has many implications. From the point of view of understanding human genetic disease, it means that we have to face the likelihood that susceptibility to a disease can be influenced by variants at many loci, each with small effects (Kryukov et al. 2007). The pervasive presence of deleterious mutations throughout the genome contributes to inbreeding depression (Charlesworth and Willis 2009) and may mean that the effective population size is reduced by background selection effects, even in regions of the genome with normal levels of genetic recombination (Loewe and Charlesworth 2007). Their presence may contribute so strongly to Hill–Robertson effects (Hill and Robertson 1966; Felsenstein 1974) that they cause severely reduced levels of diversity and adaptation in low-recombination regions of the genome (Charlesworth et al. 2010) and create a selective advantage to maintaining nonzero levels of recombination (Keightley and Otto 2006; Charlesworth et al. 2010). In addition, having an estimate of the distribution of selection coefficients against deleterious nonsynonymous mutations allows their contribution to between-species divergence to be predicted, providing a way of estimating the fraction of fixed nonsynonymous differences caused by positive selection (Loewe et al. 2006; Boyko et al. 2008; Eyre-Walker and Keightley 2009).It is thus important to collect data that shed light on the properties of selection against nonsynonymous mutations in a wide range of systems and also to compare the results from different methods of estimation, since they are subject to different sources of difficulty and biases. In a previous study, we proposed the use of a comparison between two related species with different effective population sizes for this purpose (Loewe and Charlesworth 2006; Loewe et al. 2006), using Drosophila miranda and D. pseudoobscura as material. These are well suited for this type of study, as they are closely related, live together in similar habitats, and yet have very different levels of silent nucleotide diversity, indicating different effective population sizes (N_e). This study was hampered by our inability to compare the same set of loci across the two species and by the small number of loci that could be used. We here present the results of a much larger study of DNA variation at X-linked and autosomal loci for these two species, using D. affinis as a basis for estimating divergence. We compare the results, applying the method of Loewe et al. (2006) with that of Eyre-Walker and Keightley (2009) for estimating the distribution of deleterious selection coefficients and with McDonald–Kreitman test-based methods for estimating the proportion of nonsynonymous differences fixed by positive selection. While broadly confirming the conclusions from earlier studies, we note some possible sources of bias and describe methods for minimizing their effects.     

The Origin of Spontaneous Mutation in SACCHAROMYCES CEREVISIAE

Characterization of two antimutator loci in yeast shows that both are members of the same mutagenic repair system known to be responsible for almost all induced mutation (Lawrence and Christensen 1976, 1979a,b; Prakash 1976). One of the these newly isolated antimutator mutations is an allele of rev3 (Lemontt 1971b). Two other alleles of rev3 were tested and were also found to be antimutators. Double mutants carrying rev3 and mutator mutations of rad3, rad51 or rad18 are like rev3 single mutants with respect to spontaneous mutation rate, supporting the hypothesis (Hastings, Quah and von Borstel 1976) that many mutators in yeast act by channelling spontaneous lesions from accurate to mutagenic repair. However, the enhanced mutation rate seen in a radiation-resistant mutator mutant mut1 is not dependent on REV3, but is dependent on another gene designated ANT1. An additive effect on the reduction in spontaneous mutation, seen in the ant1 rev3 double-mutant strain, leads to the conclusion that at least 90% of spontaneous mutations seen in the wild type are caused by mutagenic repair of spontaneous lesions.     

Specific Duplication and Dorsoventrally Asymmetric Expression Patterns of Cycloidea-Like Genes in Zygomorphic Species of Ranunculaceae

Floral bilateral symmetry (zygomorphy) has evolved several times independently in angiosperms from radially symmetrical (actinomorphic) ancestral states. Homologs of the Antirrhinum majus Cycloidea gene (Cyc) have been shown to control floral symmetry in diverse groups in core eudicots. In the basal eudicot family Ranunculaceae, there is a single evolutionary transition from actinomorphy to zygomorphy in the stem lineage of the tribe Delphinieae. We characterized Cyc homologs in 18 genera of Ranunculaceae, including the four genera of Delphinieae, in a sampling that represents the floral morphological diversity of this tribe, and reconstructed the evolutionary history of this gene family in Ranunculaceae. Within each of the two RanaCyL (Ranunculaceae Cycloidea-like) lineages previously identified, an additional duplication possibly predating the emergence of the Delphinieae was found, resulting in up to four gene copies in zygomorphic species. Expression analyses indicate that the RanaCyL paralogs are expressed early in floral buds and that the duration of their expression varies between species and paralog class. At most one RanaCyL paralog was expressed during the late stages of floral development in the actinomorphic species studied whereas all paralogs from the zygomorphic species were expressed, composing a species-specific identity code for perianth organs. The contrasted asymmetric patterns of expression observed in the two zygomorphic species is discussed in relation to their distinct perianth architecture.     

Rate of Adaptation in Large Sexual Populations

Adaptation often involves the acquisition of a large number of genomic changes that arise as mutations in single individuals. In asexual populations, combinations of mutations can fix only when they arise in the same lineage, but for populations in which genetic information is exchanged, beneficial mutations can arise in different individuals and be combined later. In large populations, when the product of the population size N and the total beneficial mutation rate U_b is large, many new beneficial alleles can be segregating in the population simultaneously. We calculate the rate of adaptation, v, in several models of such sexual populations and show that v is linear in NU_b only in sufficiently small populations. In large populations, v increases much more slowly as log NU_b. The prefactor of this logarithm, however, increases as the square of the recombination rate. This acceleration of adaptation by recombination implies a strong evolutionary advantage of sex.IN asexual populations, beneficial mutations arising on different genotypes compete against each other and in large populations most of the beneficial mutations are lost because they arise on mediocre genetic backgrounds or acquire further beneficial mutations less rapidly than their peers—the combined effects of clonal interference and multiple mutations (Gerrish and Lenski 1998; Desai and Fisher 2007). Exchange of genetic material between individuals allows the combination of beneficial variants that arose in different lineages and can thereby speed up the process of adaptation (Fisher 1930; Muller 1932). Indeed, most life forms engage in some form of recombination, e.g., lateral gene transfer or competence for picking up DNA in bacteria, facultative sexual reproduction in yeast and plants, or obligate sexual reproduction in most animals. Some benefits of recombination for the rate of adaptation have recently been demonstrated experimentally in Caenorhabditis reinhardtii (Colegrave 2002), Escherichia coli (Cooper 2007), and Saccharomyces cerevisiae (Goddard et al. 2005); for a review of older experiments, see Rice (2002).Yet the benefits of sex become less obvious when one considers its disadvantageous effects: recombination can separate well-adapted combinations of alleles and sexual reproduction is more costly than asexual reproduction due to resources spent for mating and, in some cases, the necessity of males. The latter—in animals often termed the twofold cost of sex—implies that sexual populations can be unstable to the invasion of asexual variants. As a result, the pros and cons of sex have been the subject of many decades of debate in the theoretical literature (Crow and Kimura 1965; Maynard Smith 1968; Felsenstein 1974; Barton 1995a; Barton and Charlesworth 1998), and several different potentially beneficial aspects of sex have been identified, including the pruning of detrimental mutations (Peck 1994; Rice 1998) and host–parasite coevolution or otherwise changing environments (Charlesworth 1993; Ladle et al. 1993; Bürger 1999; Waxman and Peck 1999; Gandon and Otto 2007; Callahan et al. 2009). In the opposite situation of relatively static populations, it has been proposed that recombination is favored in the presence of negative epistasis (Feldman et al. 1980; Kondrashov 1984, 1988)—a situation when the combined detrimental effect of two unfavorable alleles is greater than the sum of the individual effects. While this may sometimes be a significant effect, most populations, especially microbes, are likely to be under continuing selection and the benefits of sex for speeding up adaptation are likely to dominate.The Fisher–Muller hypothesis is that sex speeds up adaptation by combining beneficial variants. Moreover, it has been demonstrated by Hill and Robertson (1966) that linkage decreases the efficacy of selection. This detrimental effect of linkage, known as the “Hill–Robertson effect,” causes selection for higher recombination rates, which has been shown by analyzing recombination modifier alleles at a locus linked to two competing segregating loci (Otto and Barton 1997; Iles et al. 2003; Barton and Otto 2005; Roze and Barton 2006; Martin et al. 2006). Hitchhiking of the allele that increases the recombination rates with the sweeping linked loci results in effective selection for increased recombination.Experiments and simulation studies suggest that the Hill–Roberston effect is more pronounced and selection for recombination modifiers is stronger in large populations with many sweeping loci (Felsenstein 1974; Colegrave 2002; Iles et al. 2003). However, the quantitative understanding of the effect of recombination in large populations is limited. Rouzine and Coffin have studied the role of recombination in the context of evolution of drug resistance in human immunodeficiency virus, finding that recombination of standing variation speeds up adaptation by producing anomalously fit individuals at the high fitness edge of the distribution (Rouzine and Coffin 2005; Gheorghiu-Svirschevski et al. 2007). The effects of epistatic interactions between polymorphisms and recombination on the dynamics of selection have recently been analyzed by Neher and Shraiman (2009). Yet none of these works consider the effects of new beneficial mutations. In the absence of new mutations (and in the absence of heterozygous advantage that can maintain polymorphisms) the fitness soon saturates as most alleles become extinct and standing variation disappears. Thus the crucial point that must be addressed is the balance between selection and recombination of existing variation and the injection of additional variation by new mutations.Here, we study the dynamics of continual evolution via new mutations, selection, and recombination using several models of recombination. Our primary models most naturally apply when periods of asexual reproduction occur between matings, so that they approximate the life style of facultatively outcrossing species such as S. cerevisiae, some plants, and C. elegans, which reproduce asexually most of the time but undergo extensive recombination when outcrossing. The models enable us to study analytically the explicit dependence of the rate of adaptation and of the dynamics of the beneficial alleles on the important parameters such as the outcrossing rate and population size. In an independent study N. H. Barton and J. Coe (personal communication) calculate the rate of adaptation for obligate sexual organisms using several different multilocus models of recombination, including the free recombination model studied here. The relation of our work to theirs, as well as to that of Cohen et al. (2005, 2006) who have also studied the effects of recombination with multiple new mutations, is commented on in the discussion.When deleterious mutations can be neglected, the rate of adaptation is the product of the rate of production of favorable mutations NU_b (N being the population size and U_b the genomewide beneficial mutation rate), the magnitude of their effect, and their fixation probability. The fixation probability is dominated by the probability that the allele becomes established, i.e., that it rises to high enough numbers in the population that it is very unlikely to die out by further stochastic fluctuations. In a homogeneous population a single beneficial mutation with selective advantage s has a probability of establishment and eventual fixation of (in discrete generation models, P_e≈2s) (Moran 1959). In a heterogeneous population, however, a novel beneficial mutation can arise on different genetic backgrounds and its establishment probability will thus vary, being greater if it arises in a well-adapted individual. But even well-adapted genotypes soon fall behind due to sweeps of other beneficial mutations and combinations. To avoid extinction, descendants of the novel mutation thus have to move to fitter genetic backgrounds via recombination in outcrossing events (Rice 2002). As a result the establishment probability decreases as the rate of average fitness gain, v, in the population increases. But the rate of average fitness gain or, equivalently, the rate of adaptation itself depends on the establishment probability. These two quantities therefore have to be determined self-consistently.In this article we analyze several models via self-consistent calculations of the fixation probability of new mutations. For a given production rate of beneficial mutations NU_b, we find that interference between mutations is of minor importance if the recombination rate r exceeds . In this regime, the rate of adaption is v ≈ NU_bs² as found for sequential mutations or in the absence of linkage. At recombination rates below , however, v grows only logarithmically with log NU_b. We find this behavior in all our models and argue that it obtains more generally. The prefactor of the log NU_b increases with the square of the recombination rate, implying a strong benefit of recombination in large populations.     

Distribution of Fitness Effects Caused by Single-Nucleotide Substitutions in Bacteriophage f1

Empirical knowledge of the fitness effects of mutations is important for understanding many evolutionary processes, yet this knowledge is often hampered by several sources of measurement error and bias. Most of these problems can be solved using site-directed mutagenesis to engineer single mutations, an approach particularly suited for viruses due to their small genomes. Here, we used this technique to measure the fitness effect of 100 single-nucleotide substitutions in the bacteriophage f1, a filamentous single-strand DNA virus. We found that approximately one-fifth of all mutations are lethal. Viable ones reduced fitness by 11% on average and were accurately described by a log-normal distribution. More than 90% of synonymous substitutions were selectively neutral, while those affecting intergenic regions reduced fitness by 14% on average. Mutations leading to amino acid substitutions had an overall mean deleterious effect of 37%, which increased to 45% for those changing the amino acid polarity. Interestingly, mutations affecting early steps of the infection cycle tended to be more deleterious than those affecting late steps. Finally, we observed at least two beneficial mutations. Our results confirm that high mutational sensitivity is a general property of viruses with small genomes, including RNA and single-strand DNA viruses infecting animals, plants, and bacteria.MUTATIONAL fitness effects are relevant to many evolutionary processes. For instance, they determine the fraction of mutations that evolves neutrally (Ohta 1992), the amount of genetic variation at the mutation–selection balance (Haldane 1937), processes of fitness decay, such as Muller''s ratchet (Butcher 1995), mutational meltdown (Lynch et al. 1993), or lethal mutagenesis (Bull et al. 2007), the ability of organisms to fix beneficial mutations and evolve novel functions (Wagner 2005), or the origin of sex and recombination (Peck et al. 1997; de Visser et al. 2003). Considerable progress has been made in characterizing mutational fitness effects using model organisms or studying genetic variation in natural populations (Eyre-Walker and Keightley 2007). For instance, mutation–accumulation experiments suggest that the average effect of spontaneous deleterious mutations is 1% or lower (Kibota and Lynch 1996) in Escherichia coli, while roughly 90% of engineered gene knockouts are viable (Baba et al. 2006) and transposon insertions reduce fitness by 3% or less on average (Elena et al. 1998). In yeast, mutation–accumulation and chemical mutagenesis experiments have shown that mutations reduce fitness by 1–4% on average in diploid strains (Zeyl and de Visser 2001; Szafraniec et al. 2003; Joseph and Hall 2004). In nematodes most mutations have fitness effects lower than 1% (Keightley and Caballero 1997; Davies et al. 1999), in Drosophila the average effect of mutations ranges from 0.5 to 3.5% (Mukai et al. 1972; Ohnishi 1977; Fernández and López-Fanjul 1996; Fry et al. 1999), and, in humans, most segregating amino acid substitutions have fitness effects lower than 10% (Eyre-Walker and Keightley 2007).Although mutation–accumulation studies provide valuable information about the average effects of deleterious mutations, their power to infer the entire distribution of mutational effects, including neutral and lethal mutations, is more limited. Also, excluding bias due to selection can be problematic, and the precise location and nature of each mutation is often unknown. On the other hand, studies based on engineering mutations have been generally restricted to large deletions or insertions, which are probably infrequent in nature compared to point mutations. A direct and powerful approach that helps us to solve these difficulties consists of introducing single-nucleotide substitutions by site-directed mutagenesis. Due to their small genome sizes, viruses are excellent systems for achieving this goal. In previous work, this technique has been used for studying mutational fitness effects in several RNA viruses (Sanjuán et al. 2004; Carrasco et al. 2007; Domingo-Calap et al. 2009). However, less is known for DNA viruses—but see Domingo-Calap et al. (2009). Here, we use this approach to characterize the distribution of mutational fitness effects in the bacteriophage f1, an inovirus of the bacteriophage m13 clade, making two important improvements over previous work: first, the number of mutations tested is higher (100) and second, the contribution of experimental error to the observed distribution is explicitly accounted for. We show that one-fifth of single-nucleotide substitutions are lethal, while viable ones reduce fitness by 11% on average and can be described by a heavy-tail two-parameter distribution such as the log-normal. Interestingly, the fraction of beneficial mutations is unexpectedly high. We also compare the average effects of different mutation types and of mutations affecting different genes.     

The Genetic Basis of Phenotypic Adaptation II: The Distribution of Adaptive Substitutions in the Moving Optimum Model

We consider a population that adapts to a gradually changing environment. Our aim is to describe how ecological and genetic factors combine to determine the genetic basis of adaptation. Specifically, we consider the evolution of a polygenic trait that is under stabilizing selection with a moving optimum. The ecological dynamics are defined by the strength of selection, , and the speed of the optimum, ; the key genetic parameters are the mutation rate Θ and the variance of the effects of new mutations, ω. We develop analytical approximations within an “adaptive-walk” framework and describe how selection acts as a sieve that transforms a given distribution of new mutations into the distribution of adaptive substitutions. Our analytical results are complemented by individual-based simulations. We find that (i) the ecological dynamics have a strong effect on the distribution of adaptive substitutions and their impact depends largely on a single composite measure , which combines the ecological and genetic parameters; (ii) depending on γ, we can distinguish two distinct adaptive regimes: for large γ the adaptive process is mutation limited and dominated by genetic constraints, whereas for small γ it is environmentally limited and dominated by the external ecological dynamics; (iii) deviations from the adaptive-walk approximation occur for large mutation rates, when different mutant alleles interact via linkage or epistasis; and (iv) in contrast to predictions from previous models assuming constant selection, the distribution of adaptive substitutions is generally not exponential.AN important aim for both empirical and theoretical evolutionary biologists is to better understand the genetics of adaptation (e.g., Orr 2005a). For example, among the multitude of mutations that arise in a population, which ones are eventually fixed and contribute to evolutionary change? That is, given a distribution of new mutations, what is the distribution of adaptive substitutions (or fixed mutations)? Here, distribution means the probability distribution of the effects of mutations on either the phenotype or the fitness of their carriers. In principle, both the distribution of new mutations and the distribution of adaptive substitutions can be measured empirically, the former from mutation accumulation experiments (Eyre-Walker and Keightley 2007) and the latter from QTL (e.g., Bradshaw et al. 1998) or experimental evolution (Elena and Lenski 2003) studies. However, as only a small subset of all mutations is beneficial, such measurements are difficult. Therefore, a large role in studying the genetics of adaptation has to be played by theoretical modeling.In recent years, several different approaches have emerged for modeling the process of adaptation. Considerable work exists, in particular, in the context of Fisher''s geometric model (e.g., Fisher 1930; Kimura 1983; Orr 1998; Welch and Waxman 2005; Martin and Lenormand 2006), Gillespie''s mutational landscape model (e.g., Gillespie 1983, 1984; Orr 2002), various models of so-called “adaptive walks” on rugged fitness landscapes (e.g., Kauffman and Levin 1987; Kauffman 1993), and models of clonal interference in asexual populations (e.g., Gerrish and Lenski 1998; Park and Krug 2007). Together, these models have yielded several robust predictions. For example, both Fisher''s geometric model and the mutational landscape model predict that the distribution of adaptive substitutions should be approximately exponential (with respect to either phenotype or fitness) (Orr 1998, 2002, 2005a,b). This means that most substitutions have little effect, but that a significant fraction of the overall evolutionary change is due to a small number of substitutions with large effects. These results are in qualitative agreement with empirical data (Orr 2005a; Elena and Lenski 2003) and have shed new light on the classical debate about micro- vs. macromutationalism (Fisher 1930; Provine 2001).One way to look at adaptation is to view selection as a sieve that transforms the distribution of new mutations into the distribution of adaptive substitutions (Turner 1981; Orr and Betancourt 2001). This perspective emphasizes the role of environmental factors and directly leads to the question of how different selective regimes (sieves) affect the adaptive process. Yet, almost all studies to date have focused on the simplest possible ecological scenario: a population that, after a sudden change in the environment, is now under constant stabilizing selection.In reality, however, environmental change is often gradual rather than sudden (e.g., Hairston et al. 2005; Thompson 2005; Parmesan 2006; Perron et al. 2008). To account for this possibility, several authors (Bello and Waxman 2006; Collins et al. 2007; Kopp and Hermisson 2007; Sato and Waxman 2008; Kopp and Hermisson 2009) have recently turned to the so-called moving optimum model, which was originally devised in the field of quantitative genetics (e.g., Lynch et al. 1991; Lynch and Lande 1993; Bürger and Lynch 1995; Bürger 1999; Waxman and Peck 1999; Bürger and Gimelfarb 2002; Nunney 2003; Jones et al. 2004). In this model, the selectively favored value of a quantitative trait changes over time, such that the trait is under a mixture of stabilizing and directional selection. An important aspect of the moving optimum model is that it introduces an additional timescale (the timescale of environmental change), which is absent in the previous models.In a recent article (Kopp and Hermisson 2009) and a previous note (Kopp and Hermisson 2007), we have used the moving optimum model to investigate the time to fixation of a single mutation and the order in which mutations of different phenotypic effect go to fixation. However, the fastest mutations in the short term are not necessarily those that dominate evolution in the long term. The present article focuses on this long-term evolution, which can be characterized by the distribution of adaptive substitutions.     

A New Family of Ty1-copia-Like Retrotransposons Originated in the Tomato Genome by a Recent Horizontal Transfer Event

The use of β-lactam antibiotics has led to the evolution and global spread of a variety of resistance mechanisms, including β-lactamases, a group of enzymes that degrade the β-lactam ring. The evolution of increased β-lactam resistance was studied by exposing independent lineages of Salmonella typhimurium to progressive increases in cephalosporin concentration. Each lineage carried a β-lactamase gene (bla_TEM-1) that provided very low resistance. In most lineages, the initial response to selection was an amplification of the bla_TEM-1 gene copy number. Amplification was followed in some lineages by mutations (envZ, cpxA, or nmpC) that reduced expression of the uptake functions, the OmpC, OmpD, and OmpF porins. The initial resistance provided by bla_TEM-1 amplification allowed the population to expand sufficiently to realize rare secondary point mutations. Mathematical modeling showed that amplification often is likely to be the initial response because events that duplicate or further amplify a gene are much more frequent than point mutations. These models show the importance of the population size to appearance of later point mutations. Transient gene amplification is likely to be a common initial mechanism and an intermediate in stable adaptive improvement. If later point mutations (allowed by amplification) provide sufficient adaptive improvement, the amplification may be lost.THE extensive use of β-lactam antibiotics has led to the evolution and spread of many chromosomal-, plasmid-, and transposon-borne resistance mechanisms (Livermore 1995; Weldhagen 2004). Prominent among these mechanisms is a class of enzymes, β-lactamases, that hydrolyze the β-lactam ring (Ambler 1980; Poole 2004). TEM-1 β-lactamase, encoded by the bla_TEM-1 gene, hydrolyzes both penicillins and early cephalosporins (Matagne et al. 1990). As bacteria developed resistance, stable extended-spectrum cephalosporins (ESCs) were introduced, leading to evolution of TEM sequence variants with improved ESC hydrolysis (Petrosino et al. 1998). Resistance to β-lactams can also result from mutations that reduce levels of outer membrane proteins involved in uptake, altered target proteins (penicillin-binding proteins) to reduce β-lactam binding, or increased expression of efflux pumps that export the antibiotics (Poole 2004; Martínez-Martínez 2008; Zapun et al. 2008).Resistance to β-lactam antibiotics is linearly correlated with the lactamase level over a large range (Nordström et al. 1972) and resistance to β-lactam antibiotics can be provided by increasing enzyme levels. An early illustration of this process is the finding that Escherichia coli can develop ampicillin resistance by amplifying its ampC gene (Edlund and Normark 1981). Similar amplification has been observed in both eubacteria and eukaryotes (Craven and Neidle 2007; Wong et al. 2007) in response to various selective pressures, including antibiotics (Andersson and Hughes 2009; Sandegren and Andersson 2009). In an unselected bacterial population, the frequency of cells with a duplication of any specific chromosomal region ranges between 10⁻² and 10⁻⁵ depending on the region (Anderson and Roth 1981), whereas a point mutation in that gene is expected to be carried by perhaps 1 cell in 10⁷–10⁸ (Hudson et al. 2002). Thus, the rate of duplication formation is ∼10⁻⁵/cell/division and further increases ∼0.01/cell/division (Pettersson et al. 2008) while the base substitution rate is ∼10⁻¹⁰/cell/division/base pair (Hudson et al. 2002). Thus, it is apparent that variants with an increased level of any enzyme activity are more likely to owe the increase to a gene copy number change than to a point mutation. Furthermore, because of the high intrinsic instability of tandem amplifications, haploid segregants are expected to take over the population when the selection pressure is released (Pettersson et al. 2008).To examine the importance of gene amplification in bacterial adaptation to cephalosporins, several independent Salmonella typhimurium lineages carrying the bla_TEM-1 gene were allowed to develop resistance to progressively increased concentrations of cephalothin (a first-generation cephalosporin) and cefaclor (a second-generation cephalosporin). As these lineages developed resistance to higher antibiotic levels, amplification of the bla_TEM-1 gene was the primary and most common resistance mechanism, which in some cases was followed by acquisition of rare point mutations that provided stable resistance.     

Suppression of Mitochondrial DNA Instability of Autosomal Dominant Forms of Progressive External Ophthalmoplegia-Associated ANT1 Mutations in Podospora anserina

Maintenance and expression of mitochondrial DNA (mtDNA) are essential for the cell and the organism. In humans, several mutations in the adenine nucleotide translocase gene ANT1 are associated with multiple mtDNA deletions and autosomal dominant forms of progressive external ophthalmoplegia (adPEO). The mechanisms underlying the mtDNA instability are still obscure. A current hypothesis proposes that these pathogenic mutations primarily uncouple the mitochondrial inner membrane, which secondarily causes mtDNA instability. Here we show that the three adPEO-associated mutations equivalent to A114P, L98P, and V289M introduced into the Podospora anserina ANT1 ortholog dominantly cause severe growth defects, decreased reactive oxygen species production (ROS), decreased mitochondrial inner membrane potential (Δψ), and accumulation of large-scale mtDNA deletions leading to premature death. Interestingly, we show that, at least for the adPEO-type M106P and A121P mutant alleles, the associated mtDNA instability cannot be attributed only to a reduced membrane potential or to an increased ROS level since it can be suppressed without restoration of the Δψ or modification of the ROS production. Suppression of mtDNA instability due to the M106P and A121P mutations was obtained by an allele of the rmp1 gene involved in nucleo-mitochondrial cross- talk and also by an allele of the AS1 gene encoding a cytosolic ribosomal protein. In contrast, the mtDNA instability caused by the S296M mutation was not suppressed by these alleles.THE maintenance and expression of mitochondrial DNA (mtDNA) depend on many nuclear-encoded gene products. Recent studies have shown that defects in this maintenance can have devastating consequences for the cell and the organism. In humans, these defects are an important cause of neurological diseases including autosomal dominant (or recessive) progressive external ophthalmoplegia (adPEO) (Chinnery 2003; Copeland 2008). These disorders are characterized by multiple large-scale deletions of mtDNA. Three different genes that can cause PEO with multiple mtDNA deletions have been identified: the mtDNA polymerase (POLG), the heart/muscle isoform of the adenine nucleotide translocator (ANT1), and the mitochondrial DNA helicase, Twinkle.The adenine nucleotide translocator (ANT), also known as the ADP/ATP mitochondrial translocator, is the most abundant protein in the inner mitochondrial membrane (Riccio et al. 1975; Nury et al. 2006; Klingenberg 2008). It exports ATP produced by mitochondrial oxidative phosphorylation toward the cytosol to meet the energy requirements of the cell; in exchange, it transports ADP into the mitochondrial matrix to fuel the conversion of ADP to ATP by the F₁F_O-ATP synthase. In humans, four isoforms of the ANT protein exist, and they are differently expressed in a tissue-specific manner (Stepien et al. 1992; Palmieri 2004; Dolce et al. 2005). The human ANT1 isoform is predominantly expressed in skeletal and cardiac muscle, and specific ANT1 mutations are associated with adPEO characterized by mtDNA instability (Kaukonen et al. 1999, 2000; Napoli et al. 2001; Komaki et al. 2002; Siciliano et al. 2003). In mice, Ant1 knockout induces mitochondrial myopathy (Graham et al. 1997), increased H₂O₂ production, and mtDNA damage and inhibits oxidative phosphorylation (Esposito et al. 1999). Some of these mutations were introduced in the AAC2 gene of Saccharomyces cerevisiae that encodes the major ADP/ATP mitochondrial translocator isoform in this organism. Numerous and sometimes contradictory effects have been reported depending in particular on the yeast laboratory strains examined (Kaukonen et al. 2000; Chen 2002, 2004; Fontanesi et al. 2004; Palmieri et al. 2005; Wang et al. 2008b).In an attempt to better understand how these mutations affect mitochondrial DNA stability and their functional consequences on mitochondrial metabolism, we decided to introduce them in the unique ADP/ATP translocator gene of Podospora anserina, PaAnt. Like S. cerevisiae, the filamentous fungus P. anserina is an excellent system for genetic and molecular analyses. In contrast to S. cerevisiae, it is a strict multicellular aerobe that can display heteroplasmic states in which intact and rearranged mitochondrial genomes coexist. In this organism, life span is a reflection of mtDNA stability, and death is always associated with large mtDNA rearrangements. “Natural death” or aging is accompanied by large-scale reorganizations of the mtDNA whereas a nuclear-controlled premature death syndrome is accompanied by the accumulation of site-specific mtDNA deletions (Belcour et al. 1999; Silar et al. 2001 for reviews). P. anserina therefore occupies an interesting position among model systems for studying the cellular consequences of mutations in the ADP/ATP translocase gene.We show here that the mutations M106P, A121P, and S296M, equivalent to the L98P, A114P (familial), and V289M (sporadic) human mutations, severely impair the vegetative and sexual development of the fungus and are responsible for decreased ROS production and for decreased inner membrane potential (Δψ). The severity of the phenotypes differs according to the mutation. The three mutations show mtDNA instability, which leads to premature death. All these mutated traits are dominant. Interestingly, the mtDNA instability associated with the M106P and A121P mutations depends on the rmp1 gene. This gene exists under two naturally occurring alleles, rmp1-1 and rmp1-2, which control mtDNA integrity in some genetic contexts (Belcour et al. 1991; Contamine et al. 1996, 2004). When associated with the rmp1-1 allele, the M106P and A121P mutations lead to rapid mtDNA instability whereas, in the presence of the rmp1-2 allele, mtDNA instability is suppressed, and life span is considerably increased. Surprisingly, suppression is not accompanied by a restoration of the Δψ or a modification in the ROS level, demonstrating that these parameters are not sufficient to explain the M106P and A121P mtDNA instability. Mitochondrial DNA instability due to the M106P and A121P mutations is also suppressed by a mutation in the AS1 gene encoding a ribosomal protein. The suppressor effects are not observed for the S296M mutation.     

Molecular Isolation of the M Gene Suggests That a Conserved-Residue Conversion Induces the Formation of Bisexual Flowers in Cucumber Plants

Sex determination in plants involves a variety of mechanisms. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling gene M. M was identified as a previously characterized putative 1-aminocyclopropane-1-carboxylic acid synthase gene, while the m allele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.SEX determination in angiosperms, including crop plants, evolves a variety of mechanisms that involve a number of different genetic and epigenetic factors (Tanurdzic and Banks 2004). Due to its diversity in sex types and to the extensive physiological and genetic studies conducted on it, cucumber (Cucumis sativus L.; 2n = 2x = 14) is becoming a model plant for sex-determination research (Atsmon 1968; Tsao 1988; Perl-Treves 1999; Tanurdzic and Banks 2004). In cucumber plants, male and female flowers are generally produced separately in the same individual; however, certain lines also produce bisexual flowers. Preliminary genetic studies have indicated that three major genes are responsible for sex expression and segregation in the cucumber plantF/f, M/m, and A/a. The F gene may promote femaleness, while the m gene regulates the appearance of hermaphroditic flowers on the plant. Furthermore, in combination with the homozygous recessive f gene, the recessive a gene can intensify the androecious nature (Galun 1961; Robinson et al. 1976).Sex expression in cucumber plants can also be modified by various environmental factors and plant hormones such as ethylene (Atsmon 1968; Takahashi et al. 1983; Takahashi and Jaffe 1984; Perl-Treves 1999; Yamasaki et al. 2005). A series of studies (Kamachi et al. 1997, 2000; Trebitsh et al. 1997; Yamasaki et al. 2003a; Mibus and Tatlioglu 2004; Knopf and Trebitsh 2006) have been conducted to investigate the F/f gene. These studies have shown that CsACS1G, which encodes a key enzyme of the ethylene-synthesis pathway, is the candidate gene for the F/f locus. However, the M/m gene has not been studied in as much detail as the F/f gene. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling M gene.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Essential Loci in Centromeric Heterochromatin of Drosophila melanogaster. I: The Right Arm of Chromosome 2

With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. We undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster. We also utilized available RNA interference lines to knock down the expression of genes in 2Rh as another approach to identifying essential genes. In total, we verified the existence of eight novel essential loci in 2Rh: CG17665, CG17683, CG17684, CG17883, CG40127, CG41265, CG42595, and Atf6. Two of these essential loci, CG41265 and CG42595, are synonymous with the previously characterized loci l(2)41Ab and unextended, respectively. The genetic and molecular analysis of the previously reported locus, l(2)41Ae, revealed that this is not a single locus, but rather it is a large region of 2Rh that extends from unextended (CG42595) to CG17665 and includes four of the novel loci uncovered here.THE term “heterochromatin” was introduced by Heitz (1928) to describe regions of mitotic chromosomes that remain condensed throughout the cell cycle, in contrast to regions of euchromatin, which condense only during cell division. Heterochromatin was later divided into two classes: constitutive and facultative heterochromatin (Brown 1966). Constitutive heterochromatin is found in large blocks near centromeres and telomeres, while facultative heterochromatin can be described as silenced euchromatin that undergoes heterochromatization at specific developmental stages. Other properties of constitutive heterochromatin include late replication in S phase, low gene density, strikingly reduced level of meiotic recombination, enrichment in transposable element sequences and highly repetitive satellite DNA sequences, and the ability to silence euchromatic gene expression in a phenomenon called position effect variegation.Approximately 30% of the Drosophila melanogaster genome consists of constitutive heterochromatin (Gatti and Pimpinelli 1992). Centromeric heterochromatin in D. melanogaster is composed of mainly middle-repeat satellite DNA sequences and clusters of transposable element sequences (Lohe et al. 1993; Pimpinelli et al. 1995). Genes that reside in the heterochromatin are scattered like islands between the satellites and clusters of transposable elements. On average, heterochromatic genes are larger than euchromatic genes, primarily due to the prevalent accumulation of transposable element sequences in their introns (Devlin et al. 1990; Biggs et al. 1994; Dimitri et al. 2003a,b; Hoskins et al. 2007). Heterochromatic genes also tend to be AT-rich compared to their euchromatic counterparts; there is some evidence suggesting that the coding sequences of heterochromatic genes evolve toward AT richness in response to being located in heterochromatin (Yasuhara et al. 2005; Díaz-Castillo and Golic 2007).Drosophila heterochromatin is vastly under-replicated in polytene chromosomes, so heterochromatic genes cannot easily be mapped through polytene analysis. However, by using Hoechst 33258 and N-chromosome banding techniques, Dimitri (1991) was successful in dividing heterochromatin in mitotic chromosomes into distinct cytological bands; this was an important step in mapping the precise location of heterochromatic genes because before this time heterochromatic genes could be mapped only relative to one another. Here we focus on further refining the previous mapping work on essential genes in the proximal heterochromatin of the right arm of the second chromosome (2Rh) in cytological region h41–h46 of D. melanogaster (Hilliker 1976; Hilliker et al. 1980; Coulthard et al. 2003; Myster et al. 2004).Early mapping studies in D. melanogaster putatively placed the light (lt) and rolled (rl) genes in, or near, chromosome 2 heterochromatin (Schultz 1936; Hannah 1951; Hessler 1958). The first large-scale mutagenesis specifically directed at finding vital loci in second chromosome heterochromatin was conducted by Hilliker (1976). Using heterochromatic deletions created by Hilliker and Holm (1975), Hilliker (1976) set out to map vital loci using the mutagen ethyl methanesulfonate (EMS). He identified seven individual lethal complementation groups in 2Rh that were interpreted as representing seven vital loci. One of these heterochromatic loci was identified as the previously described rl gene. Two of the remaining vital loci have since been identified: Nipped-A is synonymous with the l(2) 41Ah complementation group (Rollins et al. 1999) and RpL38 is synonymous with Minute(2)41A and Hilliker''s (1976) l(2)41Af complementation group (Marygold et al. 2005; also referred to as l(2)Ag in FlyBase). In addition, Rollins et al. (1999) found the Nipped-B gene to be located in 2Rh, but how this locus fit into the data from Hilliker (1976) was unclear.With the limited release of some of the more distal heterochromatic sequences (Hoskins et al. 2002), a more recent mutagenesis screen focusing on distal 2Rh was conducted by Myster et al. (2004). In the region defined by the overlap between Df(2R)41A8 and Df(2R)41A10 (the latter was previously shown to be deficient for most of 2Rh; Hilliker and Holm 1975), Myster et al. (2004) reported the existence of 15 vital loci, considerably more than the 4 essential loci predicted by Hilliker (1976). The discrepancy between these two studies was the catalyst for this current work. Each group used the same mutagen, EMS, yet each group came up with very different interpretations of the number of vital loci.Hilliker''s interpretation relied on earlier evidence that EMS preferentially produced point mutations and not large-scale aberrations (Lim and Snyder 1974). Assuming that the mutants isolated in his study were point mutations, or small aberrations limited to one locus, Hilliker found that some of the loci that he identified exhibited complex interallelic complementation; the most complex complementation pattern was observed with locus l(2)41Ae. On the other hand, the interpretation of Myster et al. (2004) was that heterochromatin was more sensitive to EMS and that EMS could produce large heterochromatic deletions; they proposed that the complex interallelic complementation in l(2)41Ae was due to the presence of deletions and that l(2)41Ae represented a region of 2Rh containing many genes, rather than being a single locus.To resolve these different interpretations of the genomic segment containing l(2)41Ae (i.e., is it a single locus or a region of 2Rh), we set out to map l(2)41Ae and the region surrounding the presumed location of l(2)41Ae (as in Myster et al. 2004) by performing a large-scale inter se complementation analysis between all available mutant lines that were previously mapped to l(2)41Ae (including Nipped-B). In addition, we undertook a molecular mapping and sequence analysis, using a candidate gene approach with the most recent annotation of 2Rh (Hoskins et al. 2007), to characterize the region and identify as many essential genes as possible. We also used these approaches to map l(2)41Ab and unextended [two of the more proximal complementation groups identified by Hilliker (1976)]. Finally, we also utilized available RNA interference (RNAi) lines to knock down the expression of 12 genes in 2Rh in an attempt to identify essential genes.     

Translocation and Assembly of Mitochondrially Coded Saccharomyces cerevisiae Cytochrome c Oxidase Subunit Cox2 by Oxa1 and Yme1 in the Absence of Cox18

Members of the Oxa1/YidC/Alb3 family of protein translocases are essential for assembly of energy-transducing membrane complexes. In Saccharomyces cerevisiae, Oxa1 and its paralog, Cox18, are required for assembly of Cox2, a mitochondrially encoded subunit of cytochrome c oxidase. Oxa1 is known to be required for cotranslational export of the Cox2 N-terminal domain across the inner mitochondrial membrane, while Cox18 is known to be required for post-translational export of the Cox2 C-tail domain. We find that overexpression of Oxa1 does not compensate for the absence of Cox18 at the level of respiratory growth. However, it does promote some translocation of the Cox2 C-tail domain across the inner membrane and causes increased accumulation of Cox2, which remains unassembled. This result suggests that Cox18 not only translocates the C-tail, but also must deliver it in a distinct state competent for cytochrome oxidase assembly. We identified respiring mutants from a cox18Δ strain overexpressing OXA1, whose respiratory growth requires overexpression of OXA1. The recessive nuclear mutations allow some assembly of Cox2 into cytochrome c oxidase. After failing to identify these mutations by methods based on transformation, we successfully located them to MGR1 and MGR3 by comparative hybridization to whole-genome tiling arrays and microarray-assisted bulk segregant analysis followed by linkage mapping. While Mgr1 and Mgr3 are known to associate with the Yme1 mitochondrial inner membrane i-AAA protease and to participate in membrane protein degradation, their absence does not appear to stabilize Cox2 under these conditions. Instead, Yme1 probably chaperones the folding and/or assembly of Oxa1-exported Cox2 in the absence of Mrg1 or Mgr3, since respiratory growth and cytochrome c oxidase assembly in a cox18 mgr3 double-mutant strain overexpressing OXA1 is YME1 dependent.CYTOCHROME c oxidase is the last enzyme in the pathway of aerobic respiration. Its catalytic core consists of the three largest subunits, Cox1, Cox2, and Cox3, which are encoded in mitochondrial DNA (mtDNA) in fungi and animals, and surrounded by nuclear gene products. The synthesis of these subunits and the assembly of active cytochrome oxidase is a highly complex process that requires the action of at least 30 nuclear genes in Saccharomyces cerevisiae (reviewed in Barrientos et al. 2002; Herrmann and Funes 2005; Cobine et al. 2006; Fontanesi et al. 2008). For example, functional expression of the mitochondrial COX2 gene specifically requires, at least, Pet111 to activate mRNA translation; Oxa1 for translocation of the N-terminal domain through the inner membrane; Cox20 to chaperone the processing of the Cox2 leader peptide by the inner membrane protease (Imp1, Imp2, and Som1); Cox18, Mss2, and Pnt1 to translocate the Cox2 C-terminal domain; and Sco1 and Cox17 to insert copper into the CuA site in the C-terminal domain. These functions generate a mature protein with two transmembrane helices in the inner membrane and N- and C-tail domains in the intermembrane space (IMS) that is assembled into the complex in steps involving additional factors.Oxa1 is the founding member of the Oxa1/YidC/Alb3 family of integral membrane proteins that facilitate the insertion of respiratory and energy-transducing complexes into bacterial, mitochondrial, and thylakoid membranes through protein translocase and membrane insertase activities (reviewed in Bonnefoy et al. 2009). Mitochondria of fungi, animals, and plants contain both Oxa1 proteins and paralogously related Cox18 (also known as Oxa2) proteins (Funes et al. 2004; Gaisne and Bonnefoy 2006). These proteins, and bacterial YidC proteins, share similar core topologies with five transmembrane domains. Oxa1 has a large C-terminal domain facing the matrix that interacts with mitochondrial ribosomes (Jia et al. 2003; Szyrach et al. 2003). Bacterial YidC and mitochondrial Cox18 proteins lack this domain.In S. cerevisiae, Oxa1 and Cox18 have distinct functions in the biogenesis of Cox2. Oxa1 is required for translocation of the Cox2 N-tail domain (He and Fox 1997) via a cotranslational mechanism. It is also required for translocation of the C-tail domain, although it is unclear whether this requirement involves direct participation of Oxa1 or reflects a requirement of prior N-tail topogenesis for C-tail export (Bonnefoy et al. 2009). Yeast Oxa1 also participates in the assembly of the ATP synthase (Altamura et al. 1996; Hell et al. 2001; Jia et al. 2007). Yeast Cox18 is not required for N-tail export, but in conjunction with the peripheral inner membrane protein Mss2 and the integral membrane protein Pnt1, Cox18 is required for the export of the Cox2 C-tail post-translationally and has no other known substrate (He and Fox 1999; Broadley et al. 2001; Saracco and Fox 2002; Fiumera et al. 2007). These observations show that Oxa1 alone is not capable of translocating the Cox2 C-tail, whether or not it directly participates in that reaction.S. cerevisiae OXA1 fails to complement cox18 mutations when overexpressed in otherwise wild-type cells (Saracco and Fox 2002). Similarly, COX18 fails to complement oxa1 mutations (L. E. Elliott, H. L. Fiumera and T. D. Fox, unpublished results), confirming that these proteins have distinct functions. While the precise roles of human Oxa1 and Cox18 in human cells have not been established (Stiburek et al. 2007), expression in yeast of cDNAs encoding these human proteins does partially complement the corresponding yeast mutations (Bonnefoy et al. 1994b; Gaisne and Bonnefoy 2006). Furthermore, expression of mitochondrially targeted Escherichia coli YidC in yeast partially complements cox18 mutations but not oxa1 mutations (Preuss et al. 2005). Addition of the yeast Oxa1 C-terminal ribosome-binding domain to YidC allows it to partially complement oxa1 mutations but not cox18 mutations.If Cox2 is correctly inserted into the inner membrane but prevented from assembling into cytochrome oxidase, it is degraded by a pathway largely dependent on Yme1 (Nakai et al. 1995; Pearce and Sherman 1995; Weber et al. 1996). Yme1 is a member of a conserved family of ATP-dependent AAA proteases (reviewed in Koppen and Langer 2007), whose human ortholog functions in yeast (Shah et al. 2000). Yme1 comprises the i-AAA protease, an integral inner membrane protein whose AAA and proteolytic domains are exposed in the IMS (Leonhard et al. 1996) where they interact with exported domains of Cox2 (Graef et al. 2007). When export of the Cox2 C-tail domain is prevented by an mss2 deletion, Cox2 is instead largely degraded by the m-AAA protease (Broadley et al. 2001), an enzyme homologous to Yme1 with catalytic domains in the matrix (Leonhard et al. 1996). The AAA domain of Yme1 exhibits the chaperone-like property of binding to unfolded substrates in isolated mitochondria and in vitro, an interaction that precedes degradation (Leonhard et al. 1999; Graef et al. 2007). However, Yme1 has not previously been shown to participate as a chaperone in the productive folding of mitochondrial proteins in vivo.In this study we have examined the phenotype of a nonrespiring cox18Δ deletion strain overproducing Oxa1 from multiple plasmid-borne copies of the wild-type OXA1 gene. Surprisingly, we found that overproduced Oxa1 does support limited export of the Cox2 C-tail domain, but cytochrome oxidase is not assembled. Thus, in wild-type cells Cox18 appears not only to translocate the Cox2 C-tail, but also to do so in a fashion that promotes its proper folding and/or assembly. Respiring mutants selected from this strain inactivate either of two recently discovered adaptor subunits of the i-AAA protease. Respiratory growth of these strains remains dependent upon Yme1 activity, suggesting that under these conditions Yme1 can function as a chaperone in the assembly of Cox2 into cytochrome oxidase.     

The Genetic Signature of Conditional Expression

Conditionally expressed genes have the property that every individual in a population carries and transmits the gene, but only a fraction, φ, expresses the gene and exposes it to natural selection. We show that a consequence of this pattern of inheritance and expression is a weakening of the strength of natural selection, allowing deleterious mutations to accumulate within and between species and inhibiting the spread of beneficial mutations. We extend previous theory to show that conditional expression in space and time have approximately equivalent effects on relaxing the strength of selection and that the effect holds in a spatially heterogeneous environment even with low migration rates among patches. We support our analytical approximations with computer simulations and delineate the parameter range under which the approximations fail. We model the effects of conditional expression on sequence polymorphism at mutation–selection–drift equilibrium, allowing for neutral sites, and show that sequence variation within and between species is inflated by conditional expression, with the effect being strongest in populations with large effective size. As φ decreases, more sites are recruited into neutrality, leading to pseudogenization and increased drift load. Mutation accumulation diminishes the degree of adaptation of conditionally expressed genes to rare environments, and the mutational cost of phenotypic plasticity, which we quantify as the plasticity load, is greater for more rarely expressed genes. Our theory connects gene-level relative polymorphism and divergence with the spatial and temporal frequency of environments inducing gene expression. Our theory suggests that null hypotheses for levels of standing genetic variation and sequence divergence must be corrected to account for the frequency of expression of the genes under study.IN genetically and ecologically subdivided populations, some individuals will experience a local environment very different from others, making it difficult to evolve a single adaptation adequate for all local conditions. Phenotypic plasticity allows organisms to respond adaptively to spatially and temporally varying environments by developing alternative phenotypes that enhance fitness under local conditions (Scheiner 1993; Via et al. 1995). Examples of alternative phenotypes, i.e., polyphenisms, include the defensive morphologies in Daphnia and algae induced by the presence of predators (e.g., Lively 1986; DeWitt 1998; Harvell 1998; Hazel et al. 2004); the winged and wingless morphs of bean beetles responding to resource variation (e.g., Abouheif and Wray 2002; Roff and Gelinas 2003; Lommen et al. 2005); and bacterial genes involved in traits such as quorum sensing, antibiotic production, biofilm formation, and virulence (Fuqua et al. 1996). The developmental basis of such alternative phenotypes often lies in the inducible expression of some genes in some individuals by environmental variables. That is, all individuals carry and transmit the conditionally expressed genes but only a fraction of individuals, φ, express them when environmental conditions are appropriate.The genes underlying plastic traits should experience relaxed selection due to conditional expression. Wade and co-workers have shown that genes hidden from natural selection in a fraction of individuals in the population by X-linked (Whitlock and Wade 1995; Linksvayer and Wade 2009) or sex-limited expression (Wade 1998; Demuth and Wade 2007) experience relaxed selective constraint. In Drosophila spp., sequence data for genes with maternally limited expression quantitatively support the theoretical predictions both for within-species polymorphism (Barker et al. 2005; Cruickshank and Wade 2008) and for between-species divergence (Barker Et Al 2005; Demuth and Wade 2007; Cruickshank and Wade 2008). Furthermore, male-specific genes in the facultatively sexual pea aphid have been shown to have elevated levels of sequence variation due to relaxed selection (Brisson and Nuzhdin 2008). Genes with spatially restricted expression in a heterogeneous environment should likewise experience relaxed selection. Adaptation to the most common environment in an ecologically subdivided population (Rosenzweig 1987; Holt and Gaines 1992; Holt 1996) allows deleterious mutations to accumulate in traits expressed in rare environments (Kawecki 1994; Whitlock 1996).Here we extend these results by quantifying the consequences of relaxed selection on conditionally expressed genes. Specifically, we show that, with weak selection, spatial and temporal fluctuations in selection intensity generate approximately equivalent effects on mean trait fitness, even with low rates of migration between habitats, resulting in a great simplification of analytical results. Our analytical approximations are supported with deterministic and stochastic simulations, and we note the conditions under which the approximations fail. We then derive general expressions for (1) the expected level of sequence polymorphism within populations under mutation, migration, drift, and purifying selection with conditional gene expression; (2) the rate of sequence divergence among populations, for dominant and recessive mutations; and (3) the reduction in mean population fitness due to accumulation of deleterious mutations at conditionally expressed loci. We find that the rate of accumulation of deleterious mutations for conditionally expressed genes is accelerated and the probability of fixation of beneficial mutations is reduced, causing a reduction in the fitness of conditional traits and an inflation in sequence variation within and between species. Our results suggest that evolutionary null hypotheses must be adjusted to account for the frequency of expression of genes under study, such that signatures of elevated within- or between-species sequence variation are not necessarily evidence of the action of diversifying natural selection. Furthermore, if conditional expression is due to spatial heterogeneity, we show that the level of genetic variation in a sample will often depend on whether or not genotypes were sampled from the selective habitat, the neutral habitat, or both. In the discussion we address the scope and limitations of our theory, as well as its implications for the maintenance of genetic variation, adaptive divergence between species, constraints on phenotypic plasticity, and evolutionary inference from sequence data.