PLoS Biology

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Online ISSN: 1545-7885
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Article
The evolution of cooperation is a paradox because natural selection should favor exploitative individuals that avoid paying their fair share of any costs. Such conflict between the self-interests of cooperating individuals often results in the evolution of complex, opponent-specific, social strategies and counterstrategies. However, the genetic and biological mechanisms underlying complex social strategies, and therefore the evolution of cooperative behavior, are largely unknown. To address this dearth of empirical data, we combine mathematical modeling, molecular genetic, and developmental approaches to test whether variation in the production of and response to social signals is sufficient to generate the complex partner-specific social success seen in the social amoeba Dictyostelium discoideum. Firstly, we find that the simple model of production of and response to social signals can generate the sort of apparent complex changes in social behavior seen in this system, without the need for partner recognition. Secondly, measurements of signal production and response in a mutant with a change in a single gene that leads to a shift in social behavior provide support for this model. Finally, these simple measurements of social signaling can also explain complex patterns of variation in social behavior generated by the natural genetic diversity found in isolates collected from the wild. Our studies therefore demonstrate a novel and elegantly simple underlying mechanistic basis for natural variation in complex social strategies in D. discoideum. More generally, they suggest that simple rules governing interactions between individuals can be sufficient to generate a diverse array of outcomes that appear complex and unpredictable when those rules are unknown.
 
Image: Nick D. Kim
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Article
The study of post-reproductive lifespan has been of interest primarily with regard to the extended post-menopausal lifespan seen in humans. This unusual feature of human demography has been hypothesized to have evolved because of the “grandmother” effect, or the contributions that post-reproductive females make to the fitness of their children and grandchildren. While some correlative analyses of human populations support this hypothesis, few formal, experimental studies have addressed the evolution of post-reproductive lifespan. As part of an ongoing study of life history evolution in guppies, we compared lifespans of individual guppies derived from populations that differ in their extrinsic mortality rates. Some of these populations co-occur with predators that increase mortality rate, whereas other nearby populations above barrier waterfalls are relatively free from predation. Theory predicts that such differences in extrinsic mortality will select for differences in the age at maturity, allocation of resources to reproduction, and patterns of senescence, including reproductive declines. As part of our evaluation of these predictions, we quantified differences among populations in post-reproductive lifespan. We present here the first formal, comparative study of the evolution of post-reproductive lifespan as a component of the evolution of the entire life history. Guppies that evolved with predators and that experienced high extrinsic mortality mature at an earlier age but also have longer lifespans. We divided the lifespan into three non-overlapping components: birth to age at first reproduction, age at first reproduction to age at last reproduction (reproductive lifespan), and age at last reproduction to age at death (post-reproductive lifespan). Guppies from high-predation environments live longer because they have a longer reproductive lifespan, which is the component of the life history that can make a direct contribution to individual fitness. We found no differences among populations in post-reproductive lifespan, which is as predicted since there can be no contribution of this segment of the life history to an individual's fitness. Prior work on the evolution of post-reproductive lifespan has been dominated by speculation and correlative analyses. We show here that this component of the life history is accessible to formal study as part of experiments that quantify the different segments of an individual's life history. Populations of guppies subject to different mortality pressures from predation evolved differences in total lifespan, but not in post-reproductive lifespan. Rather than showing the direct effects of selection characterizing other life-history traits, post-reproductive lifespan in these fish appears to be a random add-on at the end of the life history. These findings support the hypothesis that differences in lifespan evolving in response to selection are confined to the reproductive lifespan, or those segments of the life history that make a direct contribution to fitness. We also show, for the first time, that fish can have reproductive senescence and extended post-reproductive lifespans despite the general observation that they are capable of producing new primary oocytes throughout their lives.
 
Article
Author Summary All living organisms must regulate precisely the flow of water into and out of cells in order to maintain cell shape and integrity. Proteins of one family, the aquaporins, are found in virtually every living organism and play a major role in maintaining water homeostasis by acting as regulated water channels. Here we describe the first crystal structure of a yeast aquaporin, Aqy1, at 1.15 Å resolution, which represents the highest resolution structural data obtained to date for a membrane protein. Using this structural information, we address an outstanding biological question surrounding yeast aquaporins: what is the functional role of the amino-terminal extension that is characteristic of yeast aquaporins? Our structural data show that the amino terminus of Aqy1 fulfills a novel gate-like function by folding to form a cytoplasmic helical bundle with a tyrosine residue entering the water channel and occluding the cytoplasmic entrance. Molecular dynamics simulations and functional studies in combination with site-directed mutagenesis suggest that water flow is regulated through a combination of mechanosensitive gating and post-translational modifications such as phosphorylation. Our study therefore provides insight into a unique mechanism for the regulation of water flux in yeast.
 
Article
In response to an Essay by Johan Bolhuis and co-authors, Phillip Lieberman contends that rather than arising from a key recent innovation ("merge"), language arose by gradual evolution of ancient capabilities.
 
Article
The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11-12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of gamma-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C>A and +83897 T>A (OR = 0.81, 95% CI [0.681-0.972], p = 0.0049) and an at-risk SNP (-243 A>G) for morbid obesity (OR = 1.3, 95% CI [1.053-1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C>A and +83897 T>A haplotype (chi(2) = 7.637, p = 0.02). In the murine insulinoma cell line betaTC3, the G at-risk allele of SNP -243 A>G increased six times GAD2 promoter activity (p < 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The -243 A>G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic beta cells, we analyzed GAD65 antibody level as a marker of beta-cell activity and of insulin secretion. In the control group, -243 A>G, +61450 C>A, and +83897 T>A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T>A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of beta-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.
 
Article
Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.
 
The Exposure of Birds to Projected Environmental Change
Geographic Patterns and Projected Impact of Environmental Change (A, B) Patterns of change in land cover due to land-use and climate change by 2100. (C, D) The resulting potential impact for birds: the pattern of richness of species with projected range declines 50%. This represents the summed, current-day occurrence of qualifying species across a 0.5 8 grid. Patterns are given for the environmentally proactive ‘‘ Adapting Mosaic ’’ scenario (A, C), 
Environmental Change, Avian Biogeography, and Loss in Range Size Projected latitudinal pattern in type of global environmental change, geographic range size, species richness, and the resulting loss in geographic range size (8,750 bird species, 18 bands of latitude). Climate (cyan, on top and semitransparent) and land-use (red) changes between now and 2100 are evaluated for two scenarios: on the left, ''Adapting Mosaic'' (A, C, E), and on the right, ''Order from Strength'' (B, D, F). Top (A, B): Total area transformed (area plot, lighter color indicates overlap) and average (6 SE) current geographic range size of species per latitudinal band (point and line plot); Middle (C, D): Average proportional loss of range size (area plot, lighter color indicating overlap) and total number of bird species whose range currently overlaps at each latitudinal band (point and line plot). Bottom (E, F): Count of species with 50% of range transformed jointly by climate change or land-use change (stacked area plot, coloration indicates the proportion of range area that is transformed by each land change type). Whereas climate change leads to a significant net change of habitat in the polar and temperate regions, the small numbers of bird species that live there on average have very large geographic ranges. Thus, proportional contractions in range size there are much smaller than for the vast majority of bird species that live in the tropics and experience significant reductions in their smaller range sizes due to land-use change. The outcome are many species with significant range reduction in the tropics and subtropics, because of the coincidence of habitat conversion with areas of high species richness. This is particularly the case in the environmentally reactive ''Order from Strength'' scenario, where large areas of land are converted to agriculture. doi:10.1371/journal.pbio.0050157.g003
Article
Over the past few decades, land-use and climate change have led to substantial range contractions and species extinctions. Even more dramatic changes to global land cover are projected for this century. We used the Millennium Ecosystem Assessment scenarios to evaluate the exposure of all 8,750 land bird species to projected land-cover changes due to climate and land-use change. For this first baseline assessment, we assumed stationary geographic ranges that may overestimate actual losses in geographic range. Even under environmentally benign scenarios, at least 400 species are projected to suffer >50% range reductions by the year 2050 (over 900 by the year 2100). Although expected climate change effects at high latitudes are significant, species most at risk are predominantly narrow-ranged and endemic to the tropics, where projected range contractions are driven by anthropogenic land conversions. Most of these species are currently not recognized as imperiled. The causes, magnitude and geographic patterns of potential range loss vary across socioeconomic scenarios, but all scenarios (even the most environmentally benign ones) result in large declines of many species. Whereas climate change will severely affect biodiversity, in the near future, land-use change in tropical countries may lead to yet greater species loss. A vastly expanded reserve network in the tropics, coupled with more ambitious goals to reduce climate change, will be needed to minimize global extinctions.
 
Article
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 x C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.
 
Summary of the Four Demographic Models Considered in Each Population (A) Schematic diagram of each demographic model and its associated parameters (see Materials and Methods for details). Parameter values that match the observed data most closely for European-Americans (EA) and African-Americans (AA) are shown below the diagrams. (B) Average and 95% confidence intervals of Tajima’s D (blue bars), Fu and Li’s D* (red bars), and Fu and Li’s F* (pale yellow bars) for the observed data and each demographic model (using the parameters that most closely match the empirical data). Results from the standard neutral model (Constant) are also shown. DOI: 10.1371/journal.pbio.0020286.g002 
Demographically Robust Selection Genes in European- Americans
A Strong Signature of Positive Selection Spanning 115 kb on Chromosome 7q (A–D) Exons for EPHB6, TRPV6, TRPV5, and KEL are shown as gray vertical lines. A dashed black line indicates the boundary between EPHB6 and TRPV6 exons, which are approximately 1 kb apart. Transcriptional orientation is indicated by the arrows below exon positions. SNPs found in European-Americans and African-Americans are shown below. Noncoding, synonymous, and nonsynonymous SNPs are denoted as black, blue, and red vertical bars, respectively. The positions of three nonsynonymous SNPs in TRPV6 are shown with asterisks. For each of the resulting nonsynonymous amino acid changes, the most frequent amino acid in European-Americans is given first. The frequency of derived alleles, P D (B), sliding window plots of Tajima’s D (C), and nucleotide diversity, p (D), are shown across the entire region. Gaps in the sliding window plots indicate positions where sequence data were not obtained. In (B–D), European- and African-American data are shown in red and 
Article
Identifying regions of the human genome that have been targets of natural selection will provide important insights into human evolutionary history and may facilitate the identification of complex disease genes. Although the signature that natural selection imparts on DNA sequence variation is difficult to disentangle from the effects of neutral processes such as population demographic history, selective and demographic forces can be distinguished by analyzing multiple loci dispersed throughout the genome. We studied the molecular evolution of 132 genes by comprehensively resequencing them in 24 African-Americans and 23 European-Americans. We developed a rigorous computational approach for taking into account multiple hypothesis tests and demographic history and found that while many apparent selective events can instead be explained by demography, there is also strong evidence for positive or balancing selection at eight genes in the European-American population, but none in the African-American population. Our results suggest that the migration of modern humans out of Africa into new environments was accompanied by genetic adaptations to emergent selective forces. In addition, a region containing four contiguous genes on Chromosome 7 showed striking evidence of a recent selective sweep in European-Americans. More generally, our results have important implications for mapping genes underlying complex human diseases.
 
Article
Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth approximately 20x). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types ( approximately 94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.
 
Article
Upon starvation or overcrowding, Caenorhabditis elegans interrupts its reproductive cycle and forms a specialised larva called dauer (enduring). This process is regulated by TGF-beta and insulin-signalling pathways and is connected with the control of life span through the insulin pathway components DAF-2 and DAF-16. We found that replacing cholesterol with its methylated metabolite lophenol induced worms to form dauer larvae in the presence of food and low population density. Our data indicate that methylated sterols do not actively induce the dauer formation but rather that the reproductive growth requires a cholesterol-derived hormone that cannot be produced from methylated sterols. Using the effect of lophenol on growth, we have partially purified activity, named gamravali, which promotes the reproduction. In addition, the effect of lophenol allowed us to determine the role of sterols during dauer larva formation and longevity. In the absence of gamravali, the nuclear hormone receptor DAF-12 is activated and thereby initiates the dauer formation program. Active DAF-12 triggers in neurons the nuclear import of DAF-16, a forkhead domain transcription factor that contributes to dauer differentiation. This hormonal control of DAF-16 activation is, however, independent of insulin signalling and has no influence on life span.
 
Article
Author Summary One of the key molecules that modulate longevity in evolutionarily diverse organisms is the transcription factor DAF-16/FOXO. Despite its importance in aging and other biological processes, how DAF-16/FOXO activity is regulated in the nucleus is largely unknown. We report a new player important for aging modulation, the nematode homolog of host cell factor 1 (HCF-1), and show that it functions as a negative regulator of DAF-16. In worms, HCF-1 inactivation extends lifespan up to 40% and increases resistance to specific stress stimuli. To affect lifespan and stress response, HCF-1 requires the activity of DAF-16. We show that the HCF-1 protein is expressed in the nucleus and partners with DAF-16 in worms. Furthermore, we demonstrate that loss of HCF-1 results in elevated levels of DAF-16 at the promoters of its target genes and altered expression of a subset of DAF-16-regulated genes. We propose that HCF-1 modulates longevity and stress response by binding to DAF-16 and preventing the transcription factor from accessing its target gene promoters, thereby regulating the expression of DAF-16 target genes. As HCF-1 is highly conserved, our findings have important implications for aging and FOXO regulation in humans.
 
Article
The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor "silent" germline micronuclear genome by a process of "unscrambling" and fragmentation. The tiny macronuclear "nanochromosomes" typically encode single, protein-coding genes (a small portion, 10%, encode 2-8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.
 
Comparison of Nucleotide Sequence Differences in COI among 260 Species of North American Birds Pairwise comparisons between 437 COI sequences are separated into three categories: differences between individuals in the same species, differences between individuals in the same genus (not including intraspecific differences), and differences between individuals in the same family (not including intraspecific or intrageneric differences). DOI: 10.1371/journal.pbio.0020312.g001
NJ Tree of COI Sequences from 30 Species in Family Scolapacidae (Sandpipers and Kin) The divergent pair of clustered sequences of Tringa solitaria is highlighted. An asterisk indicates a COI sequence from GenBank. DOI: 10.1371/journal.pbio.0020312.g002
Intraspecific Compared to Interspecific COI Distances (K2P) for Individual Species For each species in which this comparison was possible ( n = 73), maximum intraspecific variation is compared to minimum interspecific congeneric difference. For illustration purposes shown here, 2.0% is chosen as a cutoff between usual values for intra- and interspecific variation. This divides the graph into four quadrants that represent different categories of species: (I) Intraspecific 
Article
Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10x average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.
 
Article
The human intestinal microbiota is essential to the health of the host and plays a role in nutrition, development, metabolism, pathogen resistance, and regulation of immune responses. Antibiotics may disrupt these coevolved interactions, leading to acute or chronic disease in some individuals. Our understanding of antibiotic-associated disturbance of the microbiota has been limited by the poor sensitivity, inadequate resolution, and significant cost of current research methods. The use of pyrosequencing technology to generate large numbers of 16S rDNA sequence tags circumvents these limitations and has been shown to reveal previously unexplored aspects of the "rare biosphere." We investigated the distal gut bacterial communities of three healthy humans before and after treatment with ciprofloxacin, obtaining more than 7,000 full-length rRNA sequences and over 900,000 pyrosequencing reads from two hypervariable regions of the rRNA gene. A companion paper in PLoS Genetics (see Huse et al., doi: 10.1371/journal.pgen.1000255) shows that the taxonomic information obtained with these methods is concordant. Pyrosequencing of the V6 and V3 variable regions identified 3,300-5,700 taxa that collectively accounted for over 99% of the variable region sequence tags that could be obtained from these samples. Ciprofloxacin treatment influenced the abundance of about a third of the bacterial taxa in the gut, decreasing the taxonomic richness, diversity, and evenness of the community. However, the magnitude of this effect varied among individuals, and some taxa showed interindividual variation in the response to ciprofloxacin. While differences of community composition between individuals were the largest source of variability between samples, we found that two unrelated individuals shared a surprising degree of community similarity. In all three individuals, the taxonomic composition of the community closely resembled its pretreatment state by 4 weeks after the end of treatment, but several taxa failed to recover within 6 months. These pervasive effects of ciprofloxacin on community composition contrast with the reports by participants of normal intestinal function and with prior assumptions of only modest effects of ciprofloxacin on the intestinal microbiota. These observations support the hypothesis of functional redundancy in the human gut microbiota. The rapid return to the pretreatment community composition is indicative of factors promoting community resilience, the nature of which deserves future investigation.
 
Article
Most diversity in animals and plants results from the modification of already existing structures. Many organ systems, for example, are permanently modified during evolution to create developmental and morphological diversity, but little is known about the evolution of the underlying developmental mechanisms. The theory of developmental systems drift proposes that the development of conserved morphological structures can involve large-scale modifications in their regulatory mechanisms. We test this hypothesis by comparing vulva induction in two genetically tractable nematodes, Caenorhabditis elegans and Pristionchus pacificus. Previous work indicated that the vulva is induced by epidermal growth factor (EGF)/RAS and WNT signaling in Caenorhabditis and Pristionchus, respectively. Here, we show that the evolution of vulva induction involves major molecular alterations and that this shift of signaling pathways involves a novel wiring of WNT signaling and the acquisition of novel domains in otherwise conserved receptors in Pristionchus vulva induction. First, Ppa-LIN-17/Frizzled acts as an antagonist of WNT signaling and suppresses the ligand Ppa-EGL-20 by ligand sequestration. Second, Ppa-LIN-18/Ryk transmits WNT signaling and requires inhibitory SH3 domain binding motifs, unknown from Cel-LIN-18/Ryk. Third, Ppa-LIN-18/Ryk signaling involves Axin and β-catenin and Ppa-axl-1/Axin is epistatic to Ppa-lin-18/Ryk. These results confirm developmental system drift as an important theory for the evolution of organ systems and they highlight the significance of protein modularity in signal transduction and the dynamics of signaling networks.
 
Article
Nervous system function requires proper development of two functional and morphological domains of neurons, axons and dendrites. Although both these domains are equally important for signal transmission, our understanding of dendrite development remains relatively poor. Here, we show that in C. elegans the Wnt ligand, LIN-44, and its Frizzled receptor, LIN-17, regulate dendrite development of the PQR oxygen sensory neuron. In lin-44 and lin-17 mutants, PQR dendrites fail to form, display stunted growth, or are misrouted. Manipulation of temporal and spatial expression of LIN-44, combined with cell-ablation experiments, indicates that this molecule is patterned during embryogenesis and acts as an attractive cue to define the site from which the dendrite emerges. Genetic interaction between lin-44 and lin-17 suggests that the LIN-44 signal is transmitted through the LIN-17 receptor, which acts cell autonomously in PQR. Furthermore, we provide evidence that LIN-17 interacts with another Wnt molecule, EGL-20, and functions in parallel to MIG-1/Frizzled in this process. Taken together, our results reveal a crucial role for Wnt and Frizzled molecules in regulating dendrite development in vivo.
 
Article
Large-scale adaptive radiations might explain the runaway success of a minority of extant vertebrate clades. This hypothesis predicts, among other things, rapid rates of morphological evolution during the early history of major groups, as lineages invade disparate ecological niches. However, few studies of adaptive radiation have included deep time data, so the links between extant diversity and major extinct radiations are unclear. The intensively studied Mesozoic dinosaur record provides a model system for such investigation, representing an ecologically diverse group that dominated terrestrial ecosystems for 170 million years. Furthermore, with 10,000 species, extant dinosaurs (birds) are the most speciose living tetrapod clade. We assembled composite trees of 614-622 Mesozoic dinosaurs/birds, and a comprehensive body mass dataset using the scaling relationship of limb bone robustness. Maximum-likelihood modelling and the node height test reveal rapid evolutionary rates and a predominance of rapid shifts among size classes in early (Triassic) dinosaurs. This indicates an early burst niche-filling pattern and contrasts with previous studies that favoured gradualistic rates. Subsequently, rates declined in most lineages, which rarely exploited new ecological niches. However, feathered maniraptoran dinosaurs (including Mesozoic birds) sustained rapid evolution from at least the Middle Jurassic, suggesting that these taxa evaded the effects of niche saturation. This indicates that a long evolutionary history of continuing ecological innovation paved the way for a second great radiation of dinosaurs, in birds. We therefore demonstrate links between the predominantly extinct deep time adaptive radiation of non-avian dinosaurs and the phenomenal diversification of birds, via continuing rapid rates of evolution along the phylogenetic stem lineage. This raises the possibility that the uneven distribution of biodiversity results not just from large-scale extrapolation of the process of adaptive radiation in a few extant clades, but also from the maintenance of evolvability on vast time scales across the history of life, in key lineages.
 
Article
Reinforcement refers to the evolution of increased mating discrimination against heterospecific individuals in zones of geographic overlap and can be considered a final stage in the speciation process. One the factors that may affect reinforcement is the degree to which hybrid matings result in the permanent loss of genes from a species' gene pool. Matings between females of Drosophila subquinaria and males of D. recens result in high levels of offspring mortality, due to interspecific cytoplasmic incompatibility caused by Wolbachia infection of D. recens. Such hybrid inviability is not manifested in matings between D. recens females and D. subquinaria males. Here we ask whether the asymmetrical hybrid inviability is associated with a corresponding asymmetry in the level of reinforcement. The geographic ranges of D. recens and D. subquinaria were found to overlap across a broad belt of boreal forest in central Canada. Females of D. subquinaria from the zone of sympatry exhibit much stronger levels of discrimination against males of D. recens than do females from allopatric populations. In contrast, such reproductive character displacement is not evident in D. recens, consistent with the expected effects of unidirectional cytoplasmic incompatibility. Furthermore, there is substantial behavioral isolation within D. subquinaria, because females from populations sympatric with D. recens discriminate against allopatric conspecific males, whereas females from populations allopatric with D. recens show no discrimination against any conspecific males. Patterns of general genetic differentiation among populations are not consistent with patterns of behavioral discrimination, which suggests that the behavioral isolation within D. subquinaria results from selection against mating with Wolbachia-infected D. recens. Interspecific cytoplasmic incompatibility may contribute not only to post-mating isolation, an effect already widely recognized, but also to reinforcement, particularly in the uninfected species. The resulting reproductive character displacement not only increases behavioral isolation from the Wolbachia-infected species, but may also lead to behavioral isolation between populations of the uninfected species. Given the widespread occurrence of Wolbachia among insects, it thus appears that there are multiple ways by which these endosymbionts may directly and indirectly contribute to reproductive isolation and speciation.
 
Pigmentation Patterns of a Beach Mouse (P. polionotus leucocephalus), a Mainland Mouse (P. p. subgriseus), the Resulting Hybrid (F 1 ), and the Second-Generation Reciprocal-Intercross Progeny (F 2 ) 
Location and Magnitude of QTLs Affecting Pigmentation Pattern
Genetic Architecture of Pigmentation in a Cross between Beach and Mainland Subspecies (A) Each of seven pigmentation traits for which we found significant QTLs is highlighted in a different color on a cartoon of a mouse pelt. MQM analyses showed that two LGs harbored major effect QTLs and one LG carried a minor effect QTL. For each LG, LOD scores are shown as a function of genetic distance in centimorgans (cM). Black triangles on x- axes show the position of marker loci. Each line indicates the LOD score at 5-cM intervals along the LG and are coded by colors corresponding to each of the seven traits. (B) One region of major affect maps to LG 7 and the Agouti locus maps to the peak in LOD score. (C) A second major-effect region is located on LG 1, and the Mc1r locus maps to the peak in LOD score. Both major-effect loci are statistically associated with all seven pigmentation traits studied. (D) A QTL of minor effect is located in LG 14, and the Kit locus maps near the peak. This minor effect locus is associated with four of the seven pigmentation traits. doi:10.1371/journal.pbio.0050219.g003 
Gene Expression Assays for Mc1r and Agouti in Five Pigmentation Regions in Three Peromyscus Taxa (A) Phylogenetic relationship and approximate divergence times are shown for the P. polionotus subspecies and the sister species P. maniculatus . Photos show typical pigmentation pattern for each taxon. The phylogeny and pictures together highlight the similarity in pigmentation patterns between the mainland P. polionotus and P. maniculatus, the close evolutionary relationship between P. polionotus subspecies, and the derived nature of the beach mouse phenotype. (B) Qualitative (RT-PCR) and qPCR expression levels of Mc1r and Agouti mRNA relative to beta-Actin (control gene) for five distinct tissue samples in P. polionotus subspecies ( P. p. leucocephalus [ Ppl ; white] and P. p. subgriseus [ Pps ; brown]) and P. maniculatus ( Pm ; black) are shown. A 100-bp ladder (L) flanks both sides of the RT-PCR gels. For the qPCR assays, since low Ct values indicate high expression level, we transformed the raw expression data to be more intuitive: relative expression values represent the averaged Ct values for each species subtracted from the sum of expression values across all species. Significant differences in relative expression levels between P. polionotus subspecies are indicated by asterisks (Student’s t-test, p , 0.05). Bars indicate the standard error for each assay. 
Interaction between Agouti and Mc1r Genes for One Trait, Cheek Pigmentation Black bars represent the mean phenotypic score for each Mc1r genotype when F 2 progeny were clustered by Agouti genotype; alleles are indicated by dark (D) and light (L). Phenotypic values were taken for each of 465 F 2 progeny, scored as 2 (no visible pigment), 1 (partially pigmented), and 0 (fully pigmented) as indicated by the cartoons of individual hairs. Sample size and standard error bars are provided. R 2 and p -values obtained by v 2 test are 
Article
Little is known about the genetic basis of ecologically important morphological variation such as the diverse color patterns of mammals. Here we identify genetic changes contributing to an adaptive difference in color pattern between two subspecies of oldfield mice (Peromyscus polionotus). One mainland subspecies has a cryptic dark brown dorsal coat, while a younger beach-dwelling subspecies has a lighter coat produced by natural selection for camouflage on pale coastal sand dunes. Using genome-wide linkage mapping, we identified three chromosomal regions (two of major and one of minor effect) associated with differences in pigmentation traits. Two candidate genes, the melanocortin-1 receptor (Mc1r) and its antagonist, the Agouti signaling protein (Agouti), map to independent regions that together are responsible for most of the difference in pigmentation between subspecies. A derived mutation in the coding region of Mc1r, rather than change in its expression level, contributes to light pigmentation. Conversely, beach mice have a derived increase in Agouti mRNA expression but no changes in protein sequence. These two genes also interact epistatically: the phenotypic effects of Mc1r are visible only in genetic backgrounds containing the derived Agouti allele. These results demonstrate that cryptic coloration can be based largely on a few interacting genes of major effect.
 
Article
De novo hair follicle formation in embryonic skin and new hair growth in adult skin are initiated when specialized mesenchymal dermal papilla (DP) cells send cues to multipotent epithelial stem cells. Subsequently, DP cells are enveloped by epithelial stem cell progeny and other cell types to form a niche orchestrating hair growth. Understanding the general biological principles that govern the mesenchymal-epithelial interactions within the DP niche, however, has been hampered so far by the lack of systematic approaches to dissect the complete molecular make-up of this complex tissue. Here, we take a novel multicolor labeling approach, using cell type-specific transgenic expression of red and green fluorescent proteins in combination with immunolabeling of specific antigens, to isolate pure populations of DP and four of its surrounding cell types: dermal fibroblasts, melanocytes, and two different populations of epithelial progenitors (matrix and outer root sheath cells). By defining their transcriptional profiles, we develop molecular signatures characteristic for the DP and its niche. Validating the functional importance of these signatures is a group of genes linked to hair disorders that have been largely unexplored. Additionally, the DP signature reveals novel signaling and transcription regulators that distinguish them from other cell types. The mesenchymal-epithelial signatures include key factors previously implicated in ectodermal-neural fate determination, as well as a myriad of regulators of bone morphogenetic protein signaling. These findings establish a foundation for future functional analyses of the roles of these genes in hair development. Overall, our strategy illustrates how knowledge of the genes uniquely expressed by each cell type residing in a complex niche can reveal important new insights into the biology of the tissue and its associated disease states.
 
Seymour Benzer in his office at Caltech in 1974 with a big model of Drosophila. He had a great deal of respect for an animal that not only can perform many sophisticated behaviours that humans do—such as learning, courting, and keeping time—but can also walk on the ceiling and fly.
Article
Seymour Benzer died suddenly in December 2007. This piece pays tribute to Seymour Benzer's pioneering work in solid state physics, molecular biology, and neurogenetics.
 
Article
The recent discovery of functional brown adipocytes in adult humans illuminates the potential of these cells in the treatment of obesity and its associated diseases. In rodents, brown adipocyte-like cells are known to be recruited in white adipose tissue (WAT) by cold exposure or β-adrenergic stimulation, but the molecular machinery underlying this phenomenon is not fully understood. Here, we show that inducible brown adipogenesis is mediated by the microRNA miR-196a. We found that miR-196a suppresses the expression of the white-fat gene Hoxc8 post-transcriptionally during the brown adipogenesis of white fat progenitor cells. In mice, miR-196a is induced in the WAT-progenitor cells after cold exposure or β-adrenergic stimulation. The fat-specific forced expression of miR-196a in mice induces the recruitment of brown adipocyte-like cells in WAT. The miR-196a transgenic mice exhibit enhanced energy expenditure and resistance to obesity, indicating the induced brown adipocyte-like cells are metabolically functional. Mechanistically, Hoxc8 targets and represses C/EBPβ, a master switch of brown-fat gene program, in cooperation with histone deacetylase 3 (HDAC3) through the C/EBPβ 3' regulatory sequence. Thus, miR-196a induces functional brown adipocytes in WAT through the suppression of Hoxc8, which functions as a gatekeeper of the inducible brown adipogenesis. The miR-196a-Hoxc8-C/EBPβ signaling pathway may be a therapeutic target for inducing brown adipogenesis to combat obesity and type 2 diabetes.
 
Article
The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture, while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to add crucial insight into the structural organization of an endogenous, wild-type complex.
 
Article
Author Summary Most living cells constantly renew their membrane compositions and frequently communicate with neighboring cells by delivering cargo molecules from small vesicles. A key step in cargo delivery requires the fusion of the vesicle membrane with the target membrane mediated by SNARE proteins. In most cellular compartments, fusion occurs constitutively, requiring little participation of other molecules. In other cellular compartments, such as synapses in the nervous system, vesicle fusion is predominantly triggered by intracellular calcium ions. At present, constitutive and regulated fusion modes are not well understood. In this study, we found that a mutant SNARE protein, syntaxin at the synapse, contained a building block commonly conserved for syntaxins functioning along constitutive secretory pathways. Further, our modeling predicted that the mutant syntaxin could form a tightly packed SNARE bundle closely resembling that found in the endosome, but differing from the relatively loosely packed bundle found at the wild-type synapse. Our experimental data support the hypothesis that the mutant syntaxin lowered the energy barrier for vesicle fusion by tightening the SNARE bundle. These findings reveal a novel, intrinsic structural feature of the SNARE complex that regulates vesicle fusion rate at different cellular compartments.
 
Article
The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta) has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.
 
Article
The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). To determine the role of HIF-1alpha in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1alpha knockout mice (HIF-1alpha KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1alpha KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1alpha KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1alpha KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1alpha KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function.
 
Article
Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish moonshine (mon) gene specifically disrupt both embryonic and adult hematopoiesis, resulting in severe red blood cell aplasia. We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1gamma (TIF1gamma) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1gamma mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1gamma mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1gamma functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1gamma protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates.
 
Jonathan Eisen
Article
Jonathan Eisen discusses his commitment to open access publishing, and his plans as the first Academic Editor-in-Chief of PLoS Biology.
 
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Aquaporins are a family of water and small molecule channels found in organisms ranging from bacteria to animals. One of these channels, the E. coli protein aquaporin Z (AqpZ), has been shown to selectively conduct only water at high rates. We have expressed, purified, crystallized, and solved the X-ray structure of AqpZ. The 2.5 A resolution structure of AqpZ suggests aquaporin selectivity results both from a steric mechanism due to pore size and from specific amino acid substitutions that regulate the preference for a hydrophobic or hydrophilic substrate. This structure provides direct evidence on the molecular mechanisms of specificity between water and glycerol in this family of channels from a single species. It is to our knowledge the first atomic resolution structure of a recombinant aquaporin and so provides a platform for combined genetic, mutational, functional, and structural determinations of the mechanisms of aquaporins and, more generally, the assembly of multimeric membrane proteins.
 
Article
The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of
 
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Engaging the public as architects, rather than simply as subjects or objects, of the substantive content of interactive dialogue may help fulfill the democratic potential of public engagement.
 
About the Author. Carl Safina holds a PhD in ecology from Rutgers University and has published over 150 research papers, book chapters, books, and articles on seabird ecology, fisheries, and environmental policy. He is founding president of Blue Ocean Institute and adjunct professor in marine science and science communication at Stony Brook University. He has studied the ocean as a scientist, stood for it as an advocate, and conveyed his travels among sea creatures and fishing people in lyrical nonfiction writing. His first book, Song for the Blue Ocean, was chosen as a New York Times Notable Book of the Year, a Los Angeles Times Best Nonfiction selection, and a Library Journal Best Science Book selection; it won the Lannan Literary Award for nonfiction and a MacArthur “genius” prize. Dr. Safina’s second book, Eye of the Albatross, won the John Burroughs Medal for the year’s best book about the natural world and was chosen by the National Academies of Science, Engineering and Medicine as the “Year’s Best Book for Communicating Science.” The New York Times chose his Voyage of the Turtle as an Editor’s Choice. His first children’s book was published in 2010. The View from Lazy Point recently won him a special Guggenheim Fellowship and was also selected a New York Times Editor’s Choice. His book about the oil blowout in the Gulf of Mexico is A Sea in Flames (Crown Publishers, 2011). Dr. Safina hosts Saving the Ocean on PBS television.
Article
NoNo profile of events can capture all the facts, the chaos, and the many thousands of pages devoted to what the Gulf of Mexico oil blowout was—and was not. About the Author. Carl Safina holds a PhD in ecology from Rutgers University and has published over 150 research papers, book chapters, books, and articles on seabird ecology, fisheries, and environmental policy. He is founding president of Blue Ocean ... During several visits to the Gulf region in 2010 and in the months I spent writing a book on the subject, the best way I found to make sense of the blowout's many facets was as conceptual topography, its contours shaped by the interlaced factual and emotional features of the event. Perceptions were important drivers of the effects of the event. Economic effects largely reflected perceptions by tourists and seafood consumers, and psychological effects resulted from deep uncertainty over ecological effects and consequently the future viability of fishing and tourism. It seemed an event unfolded in three acts: First, the factors leading up to the blowout. Two, the varied responses during the blowout while oil was still streaming from the well. Third, the post-leak period when assessment, study, and comparison merged the technological, political, emotional, and scientific components that comprised the event [1]. It is tempting to jump from the blowout to a discussion of America's energy needs and the world's energy future. Those larger implications can seem to be the main messages of the blowout. But those messages exist independent of the blowout. That's why I will resist that connection until we discuss the blowout itself.
 
Framework Linkage Scan Framework linkage scan using 1,100 microsatellite markers for the severe (black line), moderate (red line), hip (green line), and spine (blue line) pedigree sets. The LOD score is on the y axis and the distance from the pter in Kosambi cM is on the x axis. Note that the LOD score scales are the same for all chromosomes except Chromosome 20. DOI: 10.1371/journal/pbio.0000069.g001 
Genome-Wide Scan Results and Fine-Mapping on Chromosome 20p12
The Chromosome 20p12 Linkage Region (A) Chromosome 20 linkage scan for the narrower definition of osteoporosis (severe). The LOD score is on the y axis and the distance from pter in Kosambi cM is on the x axis. (B) Region under the linkage peak is shown in more detail, including location of microsatellite markers (STRs), location of SNPs, and location of genes in the region and their direction of transcription. The legend bar indicates the distance corresponding to 100 kb. DOI: 10.1371/journal/pbio.0000069.g002 
Association Results for BMP2 SNP Haplotypes and Ser37Ala Missense Variant}
BMP2 Haplotype Region and LD (A) The location of haplotypes, markers, and the Ser37Ala missense variant is shown in relation to the location of BMP2 exons and to the LD blocks in (B). (B) Two measures of LD are shown: D9 values on the upper-left side of the plot and p values on the lower-right side. Scales for the LD strength are provided for both measures to the right. This graph shows uniformly distributed SNPs in a 370 kb region in and around the BMP2 gene. The BMP2 block and the next block downstream represent 53 kb. DOI: 10.1371/journal/pbio.0000069.g004
Article
Osteoporotic fractures are a major cause of morbidity and mortality in ageing populations. Osteoporosis, defined as low bone mineral density (BMD) and associated fractures, have significant genetic components that are largely unknown. Linkage analysis in a large number of extended osteoporosis families in Iceland, using a phenotype that combines osteoporotic fractures and BMD measurements, showed linkage to Chromosome 20p12.3 (multipoint allele-sharing LOD, 5.10; p value, 6.3 x 10(-7)), results that are statistically significant after adjusting for the number of phenotypes tested and the genome-wide search. A follow-up association analysis using closely spaced polymorphic markers was performed. Three variants in the bone morphogenetic protein 2 (BMP2) gene, a missense polymorphism and two anonymous single nucleotide polymorphism haplotypes, were determined to be associated with osteoporosis in the Icelandic patients. The association is seen with many definitions of an osteoporotic phenotype, including osteoporotic fractures as well as low BMD, both before and after menopause. A replication study with a Danish cohort of postmenopausal women was conducted to confirm the contribution of the three identified variants. In conclusion, we find that a region on the short arm of Chromosome 20 contains a gene or genes that appear to be a major risk factor for osteoporosis and osteoporotic fractures, and our evidence supports the view that BMP2 is at least one of these genes.
 
Article
The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.
 
Article
Ongoing greenhouse gas emissions can modify climate processes and induce shifts in ocean temperature, pH, oxygen concentration, and productivity, which in turn could alter biological and social systems. Here, we provide a synoptic global assessment of the simultaneous changes in future ocean biogeochemical variables over marine biota and their broader implications for people. We analyzed modern Earth System Models forced by greenhouse gas concentration pathways until 2100 and showed that the entire world's ocean surface will be simultaneously impacted by varying intensities of ocean warming, acidification, oxygen depletion, or shortfalls in productivity. In contrast, only a small fraction of the world's ocean surface, mostly in polar regions, will experience increased oxygenation and productivity, while almost nowhere will there be ocean cooling or pH elevation. We compiled the global distribution of 32 marine habitats and biodiversity hotspots and found that they would all experience simultaneous exposure to changes in multiple biogeochemical variables. This superposition highlights the high risk for synergistic ecosystem responses, the suite of physiological adaptations needed to cope with future climate change, and the potential for reorganization of global biodiversity patterns. If co-occurring biogeochemical changes influence the delivery of ocean goods and services, then they could also have a considerable effect on human welfare. Approximately 470 to 870 million of the poorest people in the world rely heavily on the ocean for food, jobs, and revenues and live in countries that will be most affected by simultaneous changes in ocean biogeochemistry. These results highlight the high risk of degradation of marine ecosystems and associated human hardship expected in a future following current trends in anthropogenic greenhouse gas emissions.
 
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Our students will go out into an astonishing new world of engineered genes and personal genomics, so why is the standard genetics syllabus stuck in the 1950s?
 
Evolutionary biology is being transformed by increasing access to burgeoning data on variation in genomes, organisms, and the environ- ment. All this can be connected to the Tree of Life (phylogeny), from populations to entire clades, and is enabled by new protocols and networks in biodiversity informatics. doi:10.1371/journal.pbio.1001466.g001 
Evolutionary biology is being transformed by increasing access to burgeoning data on variation in genomes, organisms, and the environment.
All this can be connected to the Tree of Life (phylogeny), from populations to entire clades, and is enabled by new protocols and networks in biodiversity informatics.
Natural history museum collections are tremendous repositories of specimens and data of many sorts, including phenotypes, tissue samples, vocal recordings, geographic distributions, parasites, and diet.
Photo by Jeremiah Trimble, Department of Ornithology, Museum of Comparative Zoology, Harvard University.
Developing genetic and evolutionary tools for taxa with an extensive fossil record will be an important means of integrating the study of evolutionary pattern and process.
Genomic sequence data for stickleback fish is now providing insight into evolutionary patterns, such as the reduction in the pelvic skeleton, manifest both in the fossil record and in extant populations [83]. Photograph courtesy Peter J. Park.
Developing genetic and evolutionary tools for taxa with an extensive fossil record will be an important means of integrating the study of evolutionary pattern and process. Genomic sequence data for stickleback fish is now providing insight into evolutionary patterns, such as the reduction in the pelvic skeleton, manifest both in the fossil record and in extant populations [83]. Photograph courtesy Peter J. Park. doi:10.1371/journal.pbio.1001466.g003 
Article
New theoretical and conceptual frameworks are required for evolutionary biology to capitalize on the wealth of data now becoming available from the study of genomes, phenotypes, and organisms - including humans - in their natural environments.
 
Carbohydrate Utilization by B. thetaiotaomicron and B. longum in the Presence or Absence of One Another (A) and (B) Average expression levels for five cecal samples are shown for genes encoding enzymes that shunt the indicated monosaccharides into the glycolytic or pentose phosphate pathways. Values obtained from mono-associated mice are shown with black bars, and from co-colonized mice with white bars. Significant differences in expression levels were determined using Student t-test; double asterisks (**) indicate p , 0.01, and triple asterisks (***) indicate p , 0.001. (C) GC-MS determination of monosaccharide composition in the ceca of germ-free (GF), B. thetaiotaomicron mono-associated (Bt), B. longum mono-associated (Bl), or B. thetaiotaomicron/B. longum co-colonized (Bt/ Bl) mice. Mean values 6 S.E. of biological triplicates are plotted. Significant changes in the abundance of a monosaccharide were identified using a one-way ANOVA followed by a Tukey post test; a single asterisk (*) indicates p , 0.05, double asterisks (**) indicate p , 0.01, and triple asterisks (***) indicate p , 0.001. DOI: 10.1371/journal.pbio.0040413.g002  
Changes in the B. thetaiotaomicron Transcriptome in Response to Co-colonization with B. animalis or L. casei (A) B. thetaiotaomicron genes that show significantly increased expression levels upon co-colonization with either of the two fermented dairy product– associated strains are shown. Selection criteria: (1) a 1.2-fold or greater difference in expression using the 90% confidence bound of the fold-change; (2) difference in signal ! 25; (3) p-value 0.025 using Student t-test; (4) transcript called ''present'' in 66% or more of the GeneChips in the co-colonized group; and (5) a median FDR less than 0.05 based on 50 permutated comparisons. (B) COG-based functional classification of genes identified in (A). Thirty-five of 53 genes with higher expression in the presence of L. casei and 30 of 58 genes with higher expression in the presence of B. animalis could be assigned to COGs. Functional categories with significant over-representation in the dataset of differentially expressed genes, compared to their representation in the B. thetaiotaomicron genome, were determined using a hypergeometric distribution; an asterisk (*) indicates p , 0.01. (C) All B. thetaiotaomicron SusC/D paralogs and glycoside hydrolases/polysaccharide lyases that show differential expression between the three colonization states are up-regulated in the presence of L. casei. Fold-differences are indicated in parenthesis and reflect average expression across three cecal samples when B. thetaiotaomicron is co-colonized with L. casei, compared to their average expression in three cecal samples obtained from B. thetaiotaomicron mono-associated animals. DOI: 10.1371/journal.pbio.0040413.g003  
Article
Probiotics are deliberately ingested preparations of live bacterial species that confer health benefits on the host. Many of these species are associated with the fermentation of dairy products. Despite their increasing use, the molecular details of the impact of various probiotic preparations on resident members of the gut microbiota and the host are generally lacking. To address this issue, we colonized germ-free mice with Bacteroides thetaiotaomicron, a prominent component of the adult human gut microbiota, and Bifidobacterium longum, a minor member but a commonly used probiotic. Simultaneous whole genome transcriptional profiling of both bacterial species in their gut habitat and of the intestinal epithelium, combined with mass-spectrometric analysis of habitat-associated carbohydrates, revealed that the presence of B. longum elicits an expansion in the diversity of polysaccharides targeted for degradation by B. thetaiotaomicron (e.g., mannose- and xylose-containing glycans), and induces host genes involved in innate immunity. Although the overall transcriptome expressed by B. thetaiotaomicron when it encounters B. longum in the cecum is dependent upon the genetic background of the mouse (as assessed by a mixed analysis of variance [ANOVA] model of co-colonization experiments performed in NMRI and C57BL/6J animals), B. thetaiotaomicron's expanded capacity to utilize polysaccharides occurs independently of host genotype, and is also observed with a fermented dairy product-associated strain, Lactobacillus casei. This gnotobiotic mouse model provides a controlled case study of how a resident symbiont and a probiotic species adapt their substrate utilization in response to one another, and illustrates both the generality and specificity of the relationship between a host, a component of its microbiota, and intentionally consumed microbial species.
 
Article
Author Summary Mitochondria have long been associated with aging and age-related diseases. Recent research has shown that a slight dampening of mitochondrial function can dramatically increase the lifespan of a wide range of organisms, suggesting that a similar mechanism likely operates in humans. The molecular basis of this observation is largely unknown, however. Uncovering the genes that allow altered mitochondrial function to impact longevity will give us important new insights into how mitochondria affect the aging process and will pave the way for future therapeutic developments aiming to improve healthy aging and to treat age-related diseases. Here, we used an RNAi screen in the genetic model organism C. elegans, a nematode worm, to uncover how altered mitochondrial function can modulate longevity. We found that in order for mitochondria to affect lifespan, they must communicate with several unique transcription factors in the nucleus. Notably, we discovered that the putative homeobox transcription factor CEH-23, which has not previously been implicated in longevity determination, is able to respond to changes in mitochondrial function and in turn causes an extension in lifespan.
 
Article
Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn's disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin(+) fibroblast-like synoviocytes and CD68(+) macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t(+) Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R(-) or CCR6(-) CD161(-)) CD4(+) memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.
 
Article
Author Summary In spite of continuous challenges from the ever-changing environment, biological systems exhibit incredible stability in their developmental and physiological processes. In addition to extrinsic variability caused by environmental fluctuations, cells face intrinsic variability arising from the inherent noise of gene expression and of other molecular processes. microRNAs, which act as post-transcriptional regulators of gene expression, are beginning to be recognized for their ability to confer robustness to biological systems by buffering the effects of noisy gene expression. Although noise often is viewed as destabilizing, some biological processes make use of noise in order to make stochastic decisions. In this paper, we describe a role for microRNAs in preventing the stochastic elimination of excess cells in the developing fly retina. After the sense organs that make up the eye have been specified, pruning of excess cells occurs through the action of the gene hid, the expression of which triggers cell death. Specific mechanisms are needed to protect specialized cells which need to be maintained to ensure that only excess cells are eliminated. We report that a pair of related microRNAs, miR-263a/b, protect sense organs during this pruning process by directly acting upon and limiting the expression of the proapoptotic gene hid. This example, illustrates a novel function for miRNAs in ensuring developmental robustness during apoptotic tissue pruning.
 
Genome-Wide Identification of H3K27me3 Regions (A) Comparison of ChIP-chip results with Arabidopsis genes (red boxes, where arrows indicate direction of transcription) that were previously shown to be H3K27me3 targets [21-27]. Genes are shown as green boxes (exons) and lines (introns), and H3K27me3 is shown as vertical light blue bars [posterior probability ranging from 0 (bottom) to 1 (top)]. Orange scale bars represent 1 kb. (B) Euchromatic chromosomal distribution of H3K27me3 regions. Top panels: the total length of repetitive sequences (y-axis, left-side scale) and number of genes per 100 kb (y-axis, right-side scale). Bottom panels: the total length of methylated DNA (y-axis, left-side scale) and H3K27me3 regions per 100 kb (y-axis, right-side scale). Arrows indicate the heterochromatic knob on chromosome 4. doi:10.1371/journal.pbio.0050129.g001
Characteristics of H3K27me3 Target Genes (A) Expression level of H3K27me3 target genes (red) compared to all genes (green) or genes that are not targeted by H3K27me3 (blue). Expression levels were obtained by averaging appropriate developmental stages from a recently published dataset [35] (see Materials and Methods). x- axis: expression level (log 2 scale, vertical bars indicate the bins used.); y- axis: percentage of genes with given expression level. (B) Tissue specificity of H3K27me3 target genes (red), non-H3K27me3 target genes (blue), and all genes (green), measured by entropy values (low entropy values 1⁄4 high tissue specificity). x- axis: entropy values (vertical bars indicate the bins used); y axis: fraction of genes with the given entropy value. (C) Cluster analysis of H3K27me3 target genes. H3K27me3 target genes are grouped into eight mutually exclusive clusters based on their expression patterns. Three clusters that are specifically expressed in roots (left), seeds/siliques (middle), and floral organs (right) are shown as here as examples. Other clusters are shown in Figure S3. Each row represents a gene, and each column represents a tissue type. Red or green indicate tissues in which a particular gene is highly expressed or repressed, respectively. Results of GO analyses for each cluster are shown below. The ‘‘ % in genome ’’ and ‘‘ % in cluster ’’ columns indicate the percentages of genes in the genome or in the clusters with corresponding GO terms. doi:10.1371/journal.pbio.0050129.g002 
Characteristics of H3K27me3 Regions in Arabidopsis (A) Length distribution of H3K27me3 regions [yellow: maximal gap ¼ 200 bp (MG200); brown: maximal gap ¼ 1,000 bp (MG1000); see Materials and Methods for details]. x-axis: length of H3K27me3 regions (log 10 scale); y-axis: percentage of H3K27me3 regions with corresponding length. (B) Gene-level distribution of H3K27me3. Each gene (thick horizontal bar, shown as 2.5 kb, which is the average length of Arabidopsis genes) was divided into 20 intervals (5% each interval), and the 1-kb regions upstream and downstream of each gene (thin horizontal bars) were divided into 50-bp intervals. The percentage of genes with H3K27me3 in each interval was graphed (y-axis). Dotted line indicates the percentage of the Arabidopsis genome that is associated with H3K27me3 (;5.7%). (C) The number of genes covered by each H3K27me3 region (yellow: maximal gap ¼ 200 bp; brown: maximal gap ¼ 1,000 bp). x-axis: number of genes covered by a H3K27me3 region; y-axis: percentage of H3K27me3 regions covering a given number of genes. (D) Correlation of the expression patterns of neighboring H3K27me3 target genes (maximal gap ¼ 1,000 bp) covered by the same H3K27me3 region (brown). All possible pair-wise correlations between H3K27me3 target genes were used as a control (gray). x-axis: correlation of expression patterns (1 ¼ perfect positive correlation; À1 ¼ perfect negative correlation; 0 ¼ not correlated). y-axis: percentage of gene pairs with corresponding correlation. doi:10.1371/journal.pbio.0050129.g003
LND Regions in Arabidopsis (A) A region of chromosome 1, showing the presence of LND regions (yellow vertical line) in promoters. Genes are shown as in Figure 1A. Red boxes indicate individual genes. Red arrows indicate the direction of transcription. (B) Euchromatic chromosomal distribution of LND regions shown as the total length of LND regions per 100 kb (left y-axis). Arrow indicates the heterochromatic knob on chromosome 4. Distribution of DNA methylation is shown for comparison (right y-axis). (C) Gene-level distribution of LND regions (solid lines, left y-axis) and Arabidopsis Gene Regulatory Information Server (AGRIS) transcription factor binding sites (dotted lines, right y-axis). x-axis is described as in Figure 3B. (D) Expression levels of genes with LND regions in their promoters (LND promoter genes, yellow) compared to non-LND genes (blue), or all genes (green). (E) Tissue specificity (shown as entropy) of LND promoter genes (yellow) compared to non-LND promoter genes (blue), or all genes (green). doi:10.1371/journal.pbio.0050129.g004
Relationship between H3K27me3, siRNAs, and DNA Methylation (A) The fraction of H3K27me3 regions and random control regions overlapping with siRNA clusters (blue). (B) The fraction of H3K27me3 regions and random control regions overlapping with DNA-methylated regions (blue). (C) The fractions of genes containing body DNA methylation (orange), promoter DNA methylation (red), and no DNA methylation (green) that are H3K27me3 targets. The genome averages of expressed genes are shown for comparison. doi:10.1371/journal.pbio.0050129.g005
Article
Author summary During plant and animal development, genes must be activated or repressed according to a strict temporal and spatial schedule. Histones, which are DNA-packaging proteins, play a key role in this process. For development to proceed normally, an amino acid residue (lysine 27) in histone H3 must undergo a chemical modification (called trimethylation). The modified histone (H3K27me3) maintains the repression of its target genes in appropriate tissues or developmental stages. H3K27me3 has been shown to regulate hundreds of genes and many developmental processes in animals, where it also appears to interact with other epigenetic pathways. However, the extent to which this histone modification regulates plant gene expression remained unknown. Does H3K27me3 interact with other epigenetic pathways in plants? Do plants and animals have similar H3K27me3 patterning and underlying mechanisms? To address these questions, we combined chromatin immunoprecipitation with whole-genome tiling microarrays (ChIP-chip) to identify H3K27me3-associated regions across the entire genome of the flowering plant Arabidopsis at high resolution (35 base pairs). The results suggest that H3K27me3 is a major and systematic gene silencing mechanism in plants that acts independently of small RNAs or DNA methylation. Furthermore, distinct features of Arabidopsis H3K27me3 patterning suggest that different mechanisms may be responsible for the establishment and spread of this histone modification in plants and animals.
 
Article
In this issue of PLoS Biology, Hebert et al. (2004) have set out to test the resolution and performance of “DNA barcoding,” using a single mtDNA gene, cytochrome c oxidase I (COI), for a sample of North American birds. Before turning to details of this study, it is useful as context to consider the following questions: What is DNA barcoding, and what does it promise? What is new about it? Why is it controversial? What are the potential pitfalls?
 
Decreased Catalytic Activity Is Intrinsic to the C Subunit (A) Anti-HA-tag immunoprecipitates from lysates of wild-type (wt) cells containing an empty vector (control) or wild-type, tpd3D, rrd1D/rrd2D, cdc55D/rts1D, and ppm1D cells expressing HA-tagged PPH21 were analyzed by SDS-PAGE and immunoblotting. The blots were sequentially incubated with specific antibodies against HA-tag, RRD2, and TAP42. Lanes 4-6 were not adjacent on the original blot. (B) Monomeric C subunits from the wild-type and the tpd3D strains were obtained by immunoprecipitation from lysates in which protein complexes had been disrupted by a basic pH shift, followed by neutralization (monomeric), or from control untreated lysates (complexed) (see Materials and Methods for details). The HA-PPH21 immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. (C) An aliquot of the monomeric HA-PPH21 immunoprecipitates was tested by phosphatase assays towards phosphorylase a (n ¼ 3). doi:10.1371/journal.pbio.0050155.g002
Yeast Constructs
RRD2 Interacts Physically and Functionally with TPD3 (A) HA-PPH21 or myc-RRD2 immunoprecipitates (IP) from lysates of wildtype (wt) cells co-expressing HA-PPH21 and myc-RRD2 were analyzed by SDS-PAGE and immunoblotting. The blots were sequentially incubated with anti-methyl PP2A/PPH21 (2A10), rabbit polyclonal anti-TPD3, antiRRD2, anti-TAP42, and anti-PPH21 antibody. endog., endogenous. (B) Anti-Myc-tag immunoprecipitates from lysates of wild-type cells coexpressing myc-RRD2 and HA-PPH21 were eluted by peptide competition and re-immunoprecipitated via HA-PPH21 (see Materials and Methods for details). ''Control'' indicates a wild-type strain containing both empty vectors. The re-immunoprecipitates were analyzed by SDSPAGE and immunoblotting using specific antibodies. (C) rrd1D/rrd2D and rrd1D/rrd2D/tpd3D cells containing the plasmid PYES2[myc-TPD3] were grown to log phase in complete dropout medium. Growth of the double and triple mutant was compared by spotting equal amounts of 10-fold serially diluted cells onto synthetic complete (SC) medium plates 6 1 g/l 5-fluoroorotic acid (5-FOA). The plates were incubated for 2 d (SC) or 5 d (SC þ 5-FOA) at 30 8C. Screening on 5-FOA selects for the loss of the URA3 plasmid PYES2[myc-TPD3] and indicates dependency of viability on TPD3. doi:10.1371/journal.pbio.0050155.g003
Deletion of PPE1 Restores C Subunit Catalytic Activity in the tpd3D, but Not in the rrd1D/rrd2D Strain (A) HA-tagged PPH21 was immunoprecipitated from lysates of wild-type (wt), ppe1D, rrd1D/rrd2D, rrd1D/rrd2D/ppe1D, tpd3D, and tpd3D/ppe1D cells. The immunoprecipitates were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-HA, anti-methyl PP2A, monoclonal anti-TPD3, anti-CDC55, anti-RRD2, and anti-PPE1 antibodies. (B) Aliquots of the anti–HA-tag immunoprecipitates used in (A) were assayed for phosphatase activity using phosphorylase a as substrate (n ¼ 7). (C) Logarithmically growing cultures of wild-type, ppe1D, rrd1D/rrd2D, rrd1D/rrd2D/ppe1D, tpd3D, and tpd3D/ppe1D were 10-fold serially diluted in YPD liquid medium, spotted on YPD plates, and incubated at 16 8C for 5 d, or at 30 8C or 37 8C for 3 d. doi:10.1371/journal.pbio.0050155.g005  
Article
Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Delta strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit.
 
Top-cited authors
Matthew J Phillips
  • Queensland University of Technology
Alexei J Drummond
  • University of Auckland
Jonathan A Eisen
  • University of California, Davis
Anton James Enright
  • University of Cambridge
Thomas Tuschl
  • The Rockefeller University