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The enzymes can be loaded into a nasal spray that wipes out pathogens such as Pneumococcus , Staphylococcus , and group A Strep on contact with mucous membranes. The strategy might prevent bacterial infections from spreading in close quarters like hospitals, nursing homes, and daycare centers. Fischetti says, “Clinical trials would tell us how often we had to treat, but more important, we'd have a reagent that could treat people who walk out the door of the hospital to eliminate or reduce the transmission of resistant organisms into the community. We don't have that capability right now.” | 14966545_p21 | 14966545 | Phage Therapy | 2.137116 | biomedical | Other | [
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Fischetti and his colleagues have moved on to using the enzymes systemically to wipe out Bacillus anthracis spores, preventing them from germinating and seething through the bloodstream, producing deadly toxins. An IV drip would be started after exposure to the spores. The method, Fischetti reports, is already successful in mice; clinical trials will determine how long treatment must be continued, perhaps a week or so. They have also eliminated septicemia from pneumocci with the same intravenous method. | 14966545_p22 | 14966545 | Phage Therapy | 3.47119 | biomedical | Other | [
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Up to now the enzymes must make contact with bacteria to kill, but Fischetti is hoping that a new generation of engineered enzymes will be able to kill pathogens inside cells too. A second disadvantage is that they are effective only against gram-positive bacteria, although that group includes many vicious pathogens. | 14966545_p23 | 14966545 | Phage Therapy | 2.287433 | biomedical | Other | [
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But phage enzymes seem to offer one very big advantage: resistance to them has yet to develop. Fischetti says, “We've tried very hard to identify resistant bacteria, but so far we haven't found resistant organisms in all three of the enzymes we're working with. It appears to be a very rare event, much rarer than resistance to antibiotics.” Fischetti cautions against expecting that gladsome state to last forever, but he points out that even if widespread resistance takes the same 40 or 50 years that antibiotics required to become significantly resistant, phage enzymes could buy researchers decades for inventing other approaches. | 14966545_p24 | 14966545 | Phage Therapy | 2.493047 | biomedical | Other | [
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There is no shortage of ideas for unearthing new antibiotic candidates. Why are they so slow to enter medical practice? The bottleneck, researchers agree, lies in the development process of turning them into effective therapies. Several researchers blame the big pharmaceutical companies that got so big by leading the way to new drugs for battling infectious disease, but in recent years have dropped out. Fischetti complains, “These are the big companies that have the money to develop antiinfectives, but they leave it to small biotech companies, and it's not going to happen as rapidly as it should. I think it's really unconscionable for these big companies to drop the ball because it's not going to be a billion-dollar market for them and that's what they're looking for.” | 14966545_p25 | 14966545 | Antibiotics in the 21st Century | 1.62736 | biomedical | Other | [
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Half a billion at least, says Francis Tally, a big pharmaceuticals veteran who is now chief scientific officer at Cubist Pharmaceuticals, a biotech company located in Lexington, Massachusetts. According to Tally, Cubist produced daptomycin, approved in September 2003, by licensing it from Eli Lilly, which shelved the new compound after concluding its potential market was only $250 million. | 14966545_p26 | 14966545 | Antibiotics in the 21st Century | 1.224172 | other | Other | [
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But, Tally argues, the size of the market is not the only barrier to new antibiotics. Combinatorial chemistry and the genomics revolution have simply not delivered on their early promise. “The pipeline is very dry,” he says. “There's been a real lag at the basic research level.” | 14966545_p27 | 14966545 | Antibiotics in the 21st Century | 1.613155 | biomedical | Other | [
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“Antibiotic discovery is hard,” Shapiro says. “It's a huge long process to get a decent antibiotic.” Walsh agrees. “It's easier to find inhibitors of particular enzymes for particular processes—and a very long road to convert that into something for development.” | 14966545_p28 | 14966545 | Antibiotics in the 21st Century | 1.749457 | biomedical | Other | [
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In the meantime, there is a rising clamor to slow down the rate at which bacteria develop resistance. Doctors are exhorted to cut back on prescribing antibiotics and decline to prescribe for viral diseases, which antibiotics can't combat, even when their patients badger them. | 14966545_p29 | 14966545 | Antibiotics in the 21st Century | 1.584742 | biomedical | Other | [
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But even if antibiotic consumption slowed, we will still need new antibiotics. “I always say it's not a matter of if, it's only a matter of when,” says Walsh. “There will always be a need for new antibiotics because the clock starts ticking on the useful lifetime of any antibiotic once you start to use it. That cannot be argued.” | 14966545_p30 | 14966545 | Antibiotics in the 21st Century | 1.437713 | biomedical | Other | [
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Wolbachia are intracellular gram-negative bacteria that are found in association with a variety of invertebrate species, including insects, mites, spiders, terrestrial crustaceans, and nematodes. Wolbachia are transovarialy transmitted from females to their offspring and are extremely widespread, having been found to infect 20%–75% of invertebrate species sampled . Wolbachia are members of the Rickettsiales order of the α-subdivision of the Proteobacteria phyla and belong to the Anaplasmataceae family, with members of the genera Anaplasma , Ehrlichia , Cowdria , and Neorickettsia . Six major clades (A–F) of Wolbachia have been identified to date : A, B, E, and F have been reported from insects, arachnids, and crustaceans; C and D from filarial nematodes. | 15024419_p0 | 15024419 | Introduction | 4.286119 | biomedical | Study | [
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Wolbachia– host interactions are complex and range from mutualistic to pathogenic, depending on the combination of host and Wolbachia involved. Most striking are the various forms of “reproductive parasitism” that serve to alter host reproduction in order to enhance the transmission of this maternally inherited agent. These include parthenogenesis (infected females reproducing in the absence of mating to produce infected female offspring), feminization (infected males being converted into functional phenotypic females), male-killing (infected male embryos being selectively killed), and cytoplasmic incompatibility (in its simplest form, the developmental arrest of offspring of uninfected females when mated to infected males) . | 15024419_p1 | 15024419 | Introduction | 4.16263 | biomedical | Study | [
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Wolbachia have been hypothesized to play a role in host speciation through the reproductive isolation they generate in infected hosts . They also provide an intriguing array of evolutionary solutions to the genetic conflict that arises from their uniparental inheritance. These solutions represent alternatives to classical mutualism and are often of more benefit to the symbiont than the host that is infected . From an applied perspective, it has been proposed that Wolbachia could be utilized to either suppress pest insect populations or sweep desirable traits into pest populations (e.g., the inability to transmit disease-causing pathogens) . Moreover, they may provide a new approach to the control of human and animal filariasis. Since the nematode worms that cause filariasis have an obligate symbiosis with mutualistic Wolbachia , treatment of filariasis with simple antibiotics that target Wolbachia has been shown to eliminate microfilaria production as well as ultimately killing the adult worm . | 15024419_p2 | 15024419 | Introduction | 4.245572 | biomedical | Review | [
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Despite their common occurrence and major effects on host biology, little is currently known about the molecular mechanisms that mediate the interactions between Wolbachia and their invertebrate hosts. This is partly due to the difficulty of working with an obligate intracellular organism that is difficult to culture and hard to obtain in quantity. Here we report the completion and analysis of the genome sequence of Wolbachia pipientis w Mel, a strain from the A supergroup that naturally infects Drosophila melanogaster . | 15024419_p3 | 15024419 | Introduction | 4.092011 | biomedical | Study | [
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The w Mel genome is determined to be a single circular molecule of 1,267,782 bp with a G+C content of 35.2%. This assembly is very similar to the genetic and physical map of the closely related strain w MelPop . The genome does not exhibit the GC skew pattern typical of some prokaryotic genomes that have two major shifts, one near the origin and one near the terminus of replication. Therefore, identification of a putative origin of replication and the assignment of basepair 1 were based on the location of the dnaA gene. Major features of the genome and of the annotation are summarized in Table 1 and Figure 1 . | 15024419_p4 | 15024419 | Genome Properties | 4.281169 | biomedical | Study | [
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The most striking feature of the w Mel genome is the presence of very large amounts of repetitive DNA and DNA corresponding to mobile genetic elements, which is unique for an intracellular species. In total, 714 repeats of greater than 50 bp in length, which can be divided into 158 distinct families ( Table S1 ), were identified. Most of the repeats are present in only two copies in the genome, although 39 are present in three or more copies, with the most abundant repeat being found in 89 copies. We focused our analysis on the 138 repeats of greater than 200 bp ( Table 2 ). These were divided into 19 families based upon sequence similarity to each other. These repeats were found to make up 14.2 % of the w Mel genome. Of these repeat families, 15 correspond to likely mobile elements, including seven types of insertion sequence (IS) elements, four likely retrotransposons, and four families without detectible similarity to known elements but with many hallmarks of mobile elements (flanked by inverted repeats, present in multiple copies) ( Table 2 ). One of these new elements (repeat family 8) is present in 45 copies in the genome. It is likely that many of these elements are not able to autonomously transpose since many of the transposase genes are apparently inactivated by mutations or the insertion of other transposons ( Table S2 ). However, some are apparently recently active since there are transposons inserted into at least nine genes ( Table S2 ), and the copy number of some repeats appears to be variable between Wolbachia strains (M. Riegler et al., personal communication). Thus, many of these repetitive elements may be useful markers for strain discrimination. In addition, the mobile elements likely contribute to generating the diversity of phenotypically distinct Wolbachia strains by altering or disrupting gene function ( Table S2 ). | 15024419_p5 | 15024419 | Repetitive and Mobile DNA | 4.493504 | biomedical | Study | [
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Three prophage elements are present in the genome. One is a small pyocin-like element made up of nine genes . The other two are closely related to and exhibit extensive gene order conservation with the WO phage described from Wolbachia sp. w Kue . Thus, we have named them w Mel WO-A and WO-B, based upon their location in the genome. w Mel WO-B has undergone a major rearrangement and translocation, suggesting it is inactive. Phylogenetic analysis indicates that w Mel WO-B is more closely related to the w Kue WO than to w Mel WO-A . Thus, w Mel WO-A likely represents either a separate insertion event in the Wolbachia lineage or a duplication that occurred prior to the separation of the w Mel and w Kue lineages. Phylogenetic analysis also confirms the proposed mosaic nature of the WO phage , with one block being closely related to lambdoid phage and another to P2 phage (data not shown). | 15024419_p6 | 15024419 | Repetitive and Mobile DNA | 4.324405 | biomedical | Study | [
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The irregular pattern of GC skew in w Mel is likely due in part to intragenomic rearrangements associated with the many DNA repeat elements. Comparison with a large contig from a Wolbachia species that infects Brugia malayi is consistent with this . While only translocations are seen in this plot, genetic comparisons reveal that inversions also occur between strains , which is consistent with previous studies of prokaryotic genomes that have found that the most common large-scale rearrangements are inversions that are symmetric around the origin of DNA replication . The occurrence of frequent rearrangement events during Wolbachia evolution is supported by the absence of any large-scale conserved gene order with Rickettsia genomes. The rearrangements in Wolbachia likely correspond with the introduction and massive expansion of the repeat element families that could serve as sites for intragenomic recombination, as has been shown to occur for some other bacterial species . The rearrangements in w Mel may have fitness consequences since several classes of genes often found in clusters are generally scattered throughout the w Mel genome (e.g., ABC transporter subunits, Sec secretion genes, rRNA genes, F-type ATPase genes). | 15024419_p7 | 15024419 | Genome Structure: Rearrangements, Duplications, and Deletions | 4.510075 | biomedical | Study | [
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Although the common ancestor of Wolbachia and Rickettsia likely already had a reduced, streamlined genome, w Mel has lost additional genes since that time ( Table S3 ). Many of these recent losses are of genes involved in cell envelope biogenesis in other species, including most of the machinery for producing lipopolysaccharide (LPS) components and the alanine racemase that supplies D-alanine for cell wall synthesis. In addition, some other genes that may have once been involved in this process are present in the genome, but defective and are likely in the process of being eliminated. The loss of cell envelope biogenesis genes has also occurred during the evolution of the Buchnera endosymbionts of aphids . Thus, w Mel and Buchnera have lost some of the same genes separately during their reductive evolution. Such convergence means that attempts to use gene content to infer evolutionary relatedness needs to be interpreted with caution. In addition, since Anaplasma and Ehrlichia also apparently lack genes for LPS production , it is likely that the common ancestor of Wolbachia , Ehrlichia , and Anaplasma was unable to synthesize LPS. Thus, the reports that Wolbachia -derived LPS-like compounds is involved in the immunopathology of filarial nematode disease in mammals either indicate that these Wolbachia have acquired genes for LPS synthesis or that the reported LPS-like compounds are not homologous to LPS. | 15024419_p8 | 15024419 | Genome Structure: Rearrangements, Duplications, and Deletions | 4.465195 | biomedical | Study | [
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Despite evident genome reduction in w Mel and in contrast to most small-genomed intracellular species, gene duplication appears to have continued, as over 50 gene families have apparently expanded in the w Mel lineage relative to that of all other species ( Table S4 ). Many of the pairs of duplicated genes are encoded next to each other in the genome, suggesting that they arose by tandem duplication events and may simply reflect transient duplications in evolution (deletion is common when there are tandem arrays of genes). Many others are components of mobile genetic elements, indicating that these elements have expanded significantly after entering the Wolbachia evolutionary lineage. Other duplications that could contribute to the unique biological properties of w Mel include that of the mismatch repair gene mutL (see below) and that of many hypothetical and conserved hypothetical proteins. | 15024419_p9 | 15024419 | Genome Structure: Rearrangements, Duplications, and Deletions | 4.289579 | biomedical | Study | [
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One duplication of particular interest is that of wsp , which is a standard gene for strain identification and phylogenetic reconstruction in Wolbachia . In addition to the previously described wsp , w Mel encodes two wsp paralogs , which we designate as wspB and wspC , respectively. While these paralogs are highly divergent from wsp (protein identities of 19.7% and 23.5%, respectively) and do not amplify using the standard wsp PCR primers , their presence could lead to some confusion in classification and identification of Wolbachia strains. This has apparently occurred in one study of Wolbachia strain w KueYO, for which the reported wsp gene is actually an ortholog of wspB and not an ortholog of the wsp gene. Considering that the wsp gene has been extremely informative for discriminating between strains of Wolbachia , we designed PCR primers to the w Mel wspB gene to amplify and then sequence the orthologs from the related w Ri and w AlbB Wolbachia strains from Drosophila simulans and Aedes albopictus , respectively, as well as the Wolbachia strain that infects the filarial nematode Dirofilaria immitis to determine the potential utility of this locus for strain discrimination. A comparison of genetic distances between the wsp and wspB genes for these different taxa indicates that overall the wspB gene appears to be evolving at a faster rate than wsp and, as such, may be a useful additional marker for discriminating between closely related Wolbachia strains ( Table S5 ). | 15024419_p10 | 15024419 | Genome Structure: Rearrangements, Duplications, and Deletions | 4.278307 | biomedical | Study | [
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The fraction of the genome that is repetitive DNA and the fraction that corresponds to mobile genetic elements are among the highest for any prokaryotic genome. This is particularly striking compared to the genomes of other obligate intracellular species such as Buchnera , Rickettsia , Chlamydia , and Wigglesworthia , that all have very low levels of repetitive DNA and mobile elements. The recently sequenced genomes of the intracellular pathogen Coxiella burnetti has both a streamlined genome and moderate amounts of repetitive DNA, although much less than w Mel. The paucity of repetitive DNA in these and other intracellular species is thought to be due to a combination of lack of exposure to other species, thereby limiting introduction of mobile elements, and genome streamlining . We examined the w Mel genome to try to understand the origin of the repetitive and mobile DNA and to explain why such repetitive/mobile DNA is present in w Mel, but not other streamlined intracellular species. | 15024419_p11 | 15024419 | Inefficiency of Selection in w Mel | 4.272795 | biomedical | Study | [
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We propose that the mobile DNA in w Mel was acquired some time after the separation of the Wolbachia and Rickettsia lineages but before the radiation of the Wolbachia group . The acquisition of these elements after the separation of the Wolbachia and Rickettsia lineages is suggested by the fact that most do not have any obvious homologous sequences in the genomes of other α-Proteobacteria, including the closely related Rickettsia spp. Additional evidence for some acqui-sition of foreign DNA after the Wolbachia–Rickettsia split comes from phylogenetic analysis of those genes present in w Mel, but not in the two sequenced rickettsial genomes (see Table S3 ; unpublished data). The acquisition prior to the radiation of Wolbachia is suggested by two lines of evidence. First, many of the elements are found in the genome of the distantly related Wolbachia of the nematode B. malayi . In addition, genome analysis reveals that these elements do not have significantly anomalous nucleotide composition or codon usage compared to the rest of the genome. In fact, there are only four regions of the genome with significantly anomalous composition, comprising in total only approximately 17 kbp of DNA ( Table 3 ). The lack of anomalous composition suggests either that any foreign DNA in w Mel was acquired long enough ago to allow it to “ameliorate” and become compositionally similar to endogenous Wolbachia DNA or that any foreign DNA that is present was acquired from organisms with similar composition to endogenous w Mel genes. Owing to their potential effects on genome evolution (insertional mutagenesis, catalyzing genome rearrangements), we propose that the acquisition and maintenance of these repetitive and mobile elements by w Mel have played a key role in shaping the evolution of Wolbachia . | 15024419_p12 | 15024419 | Inefficiency of Selection in w Mel | 4.557877 | biomedical | Study | [
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It is likely that much of the mobile/repetitive DNA was introduced via phage, given that three prophage elements are present; experimental studies have shown active phage in some Wolbachia and Wolbachia superinfections occur in many hosts , which would allow phage to move between strains. Whatever the mechanism of introduction, the persistence of the repetitive elements in w Mel in the face of apparently strong pressures for streamlining is intriguing. One expla-nation is that w Mel may be getting a steady infusion of mobile elements from other Wolbachia strains to counteract the elimination of elements by selection for genome streamlining. This would explain the absence of anomalous nucleotide composition of the elements. However, we believe that a major contributing factor to the presence of all the repetitive/mobile DNA in w Mel is that w Mel and possibly Wolbachia in general have general inefficiency of natural selection relative to other species. This inefficiency would limit the ability to eliminate repetitive DNA. A general inefficiency of natural selection (especially purifying selection) has been suggested previously for intracellular bacteria, based in part on observations that these bacteria have higher evolutionary rates than free-living bacteria . We also find a higher evolutionary rate for w Mel than that of the closely related intracellular Rickettsia , which themselves have higher rates than free-living α-Proteobacteria . Additionally, codon bias in w Mel appears to be driven more by mutation or drift than selection , as has been reported for Buchnera species and was suggested to be due to inefficient purifying selection . Such inefficiencies of natural selection are generally due to an increase in the relative contribution of genetic drift and mutation as compared to natural selection . Below we discuss different possible explanations for the inefficiency of selection in w Mel, especially in comparison to other intracellular bacteria. | 15024419_p13 | 15024419 | Inefficiency of Selection in w Mel | 4.539308 | biomedical | Study | [
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Low rates of recombination, such as occur in centromeres and the human Y chromosome, can lead to inefficient selection because of the linkage among genes. This has been suggested to be occurring in Buchnera species because these species do not encode homologs of RecA, which is the key protein in homologous recombination in most species . The absence of recombination in Buchnera is supported by the lack of genome rearrangements in their recent evolution . Additionally, there is apparently little or no gene flow into Buchnera strains. In contrast, w Mel encodes the necessary machinery for recombination, including RecA ( Table S6 ), and has experienced both extensive intragenomic homologous recombination and introduction of foreign DNA. Therefore, the unusual genome features of w Mel are unlikely to be due to low levels of recombination. | 15024419_p14 | 15024419 | Inefficiency of Selection in w Mel | 4.267865 | biomedical | Study | [
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] | en | 0.999997 |
Another possible explanation for inefficient selection is high mutation rates. It has been suggested that the higher evolutionary rates in intracellular bacteria are the result of high mutation rates that are in turn due to the loss of genes for DNA repair processes . This is likely not the case in w Mel since its genome encodes proteins corresponding to a broad suite of DNA repair pathways including mismatch repair, nucleotide excision repair, base excision repair, and homologous recombination ( Table S6 ). The only noteworthy DNA repair gene absent from w Mel and present in the more slowly evolving Rickettsia is mfd, which is involved in targeting DNA repair to the transcribed strand of actively transcribing genes in other species . However, this absence is unlikely to contribute significantly to the increased evolutionary rate in w Mel, since defects in mfd do not lead to large increases in mutation rates in other species . The presence of mismatch repair genes (homologs of mutS and mutL ) in w Mel is particularly relevant since this pathway is one of the key steps in regulating mutation rates in other species. In fact, w Mel is the first bacterial species to be found with two mutL homologs. Overall, examination of the predicted DNA repair capabilities of bacteria suggests that the connection between evolutionary rates in intracellular species and the loss of DNA repair processes is spurious. While many intracellular species have lost DNA repair genes in their recent evolution, different species have lost different genes and some, such as w Mel and Buchnera spp., have kept the genes that likely regulate mutation rates. In addition, some free-living species without high evolutionary rates have lost some of the same pathways lost in intracellular species, while many free-living species have lost key pathways resulting in high mutation rates . Given that intracellular species tend to have small genomes and have lost genes from every type of biological process, it is not surprising that many of them have lost DNA repair genes as well. | 15024419_p15 | 15024419 | Inefficiency of Selection in w Mel | 4.537375 | biomedical | Study | [
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] | [
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] | en | 0.999996 |
We believe that the most likely explanations for the inefficiency of selection in w Mel involve population-size related factors, such as genetic drift and the occurrence of population bottlenecks. Such factors have also been shown to likely explain the high evolutionary rates in other intracellular species . Wolbachia likely experience frequent population bottlenecks both during transovarial transmission and during cytoplasmic incompatibility mediated sweeps through host populations. The extent of these bottlenecks may be greater than in other intracellular bacteria, which would explain why w Mel has both more repetitive and mobile DNA than other such species and a higher evolutionary rate than even the related Rickettsia spp. Additional genome sequences from other Wolbachia will reveal whether this is a feature of all Wolbachia or only certain strains. | 15024419_p16 | 15024419 | Inefficiency of Selection in w Mel | 4.298988 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
There is a general consensus in the evolutionary biology literature that the mitochondria evolved from bacteria in the α-subgroup of the Proteobacteria phyla . Analysis of complete mitochondrial and bacterial genomes has very strongly supported this hypothesis . However, the exact position of the mitochondria within the α-Proteobacteria is still debated. Many studies have placed them in or near the Rickettsiales order . Some studies have further suggested that mitochondria are a sister taxa to the Rickettsia genus within the Rickettsiaceae family and thus more closely related to Rickettsia spp. than to species in the Anaplasmataceae family such as Wolbachia . | 15024419_p17 | 15024419 | Mitochondrial Evolution | 4.192296 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
In our analysis of complete genomes, including that of w Mel, the first non- Rickettsia member of the Rickettsiales order to have its genome completed, we find support for a grouping of Wolbachia and Rickettsia to the exclusion of the mitochondria, but not for placing the mitochondria within the Rickettsiales order . Specifically, phylogenetic trees of a concatenated alignment of 32 proteins show strong support with all methods (see Table S7 ) for common branching of: (i) mitochondria, (ii) Rickettsia with Wolbachia , (iii) the free-living α-Proteobacteria, and (iv) mitochondria within α-Proteobacteria. Since amino acid content bias was very severe in these datasets, protein LogDet analyses, which can correct for the bias, were also performed. In LogDet analyses of the concatenated protein alignment, both including and excluding highly biased positions, mitochondria usually branched basal to the Wolbachia–Rickettsia clade, but never specifically with Rickettsia (see Table S7 ). In addition, in phylogenetic studies of individual genes, there was no consistent phylogenetic position of mitochondrial proteins with any particular species or group within the α-Proteobacteria (see Table S8 ), although support for a specific branch uniting the two Rickettsia species with Wolbachia was quite strong. Eight of the proteins from mitochondrial genomes (YejW, SecY, Rps8, Rps2, Rps10, RpoA, Rpl15, Rpl32) do not even branch within the α-Proteobacteria, although these genes almost certainly were encoded in the ancestral mitochondrial genome . | 15024419_p18 | 15024419 | Mitochondrial Evolution | 4.263779 | biomedical | Study | [
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] | [
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This analysis of mitochondrial and α-Proteobacterial genes reinforces the view that ancient protein phylogenies are inherently prone to error, most likely because current models of phylogenetic inference do not accurately reflect the true evolutionary processes underlying the differences observed in contemporary amino acid sequences . These conflicting results regarding the precise position of mitochondria within the α-Proteobacteria can be seen in the high amount of networking in the Neighbor-Net graph of the analyses of the concatenated alignment shown in Figure 5 . An important complication in studies of mitochondrial evolution lies in identifying “α-Proteobacterial” genes for comparison . For example, in our analyses, proteins from Magnetococcus branched with other α-Proteobacterial homologs in only 17 of the 49 proteins studied, and in five cases they assumed a position basal to α-, β-, and γ-Proteobacterial homologs. | 15024419_p19 | 15024419 | Mitochondrial Evolution | 4.195308 | biomedical | Study | [
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] | [
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] | en | 0.999996 |
Many genes that were once encoded in mitochondrial genomes have been transferred into the host nuclear genomes. Searching for such genes has been complicated by the fact that many of the transfer events happened early in eukaryotic evolution and that there are frequently extreme amino acid and nucleotide composition biases in mitochondrial genomes (see above). We used the w Mel genome to search for additional possible mitochondrial-derived genes in eukaryotic nuclear genomes. Specifically, we constructed phylogenetic trees for w Mel genes that are not in either Rickettsia genomes. Five new eukaryotic genes of possible mitochondrial origin were identified: three genes involved in de novo nucleotide biosynthesis ( purD , purM , pyrD ) and two conserved hypothetical proteins . The α-Proteobacterial origin of these genes suggests that at least some of the genes of the de novo nucleotide synthesis pathway in eukaryotes might have been laterally acquired from bacteria via the mitochondria. The presence of such genes in other Proteobacteria suggests that their absence from Rickettsia is due to gene loss . This finding supports the need for additional α-Proteobacterial genomes to identify mitochondrion-derived genes in eukaryotes. | 15024419_p20 | 15024419 | Host–Symbiont Gene Transfers | 4.286983 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
While organelle to nuclear gene transfers are generally accepted, there is a great deal of controversy over whether other gene transfers have occurred from bacteria into animals. In particular, claims of transfer from bacteria into the human genome were later shown to be false . Wolbachia are excellent candidates for such transfer events since they live inside the germ cells, which would allow lateral transfers to the host to be transmitted to subsequent host generations. Consistent with this, a recent study has shown some evidence for the presence of Wolbachia- like genes in a beetle genome . The symbiosis between w Mel and D. melanogaster provides an ideal case to search for such transfers since we have the complete genomes of both the host and symbiont. Using BLASTN searches and MUMmer alignments, we did not find any examples of highly similar stretches of DNA shared between the two species. In addition, protein-level searches and phylogenetic trees did not identify any specific relationships between w Mel and D. melanogaster for any genes. Thus, at least for this host–symbiont association, we do not find any likely cases of recent gene exchange, with genes being maintained in both host and symbiont. In addition, in our phylogenetic analyses, we did not find any examples of w Mel proteins branching specifically with proteins from any invertebrate to the exclusion of other eukaryotes. Therefore, at least for the genes in w Mel, we do not find evidence for transfer of Wolbachia genes into any invertebrate genome. | 15024419_p21 | 15024419 | Host–Symbiont Gene Transfers | 4.266972 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
w Mel is predicted to have very limited capabilities for membrane transport, for substrate utilization, and for the biosynthesis of metabolic intermediates , similar to what has been seen in other intracellular symbionts and pathogens . Almost all of the identifiable uptake systems for organic nutrients in w Mel are for amino acids, including predicted transporters for proline, asparate/glutamate, and alanine. This pattern of transporters, coupled with the presence of pathways for the metabolism of the amino acids cysteine, glutamate, glutamine, proline, serine, and threonine, suggests that w Mel may obtain much of its energy from amino acids. These amino acids could also serve as material for the production of other amino acids. In contrast, carbohydrate metabolism in w Mel appears to be limited. The only pathways that appear to be complete are the tricarboxylic acid cycle, the nonoxidative pentose phosphate pathway, and glycolysis, starting with fructose-1,6-biphosphate. The limited carbohydrate metabolism is consistent with the presence of only one sugar phosphate transporter. w Mel can also apparently transport a range of inorganic ions, although two of these systems, for potassium uptake and sodium ion/proton exchange, are frameshifted. In the latter case, two other sodium ion/proton exchangers may be able to compensate for this defect. | 15024419_p22 | 15024419 | Metabolism and Transport | 4.616837 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
Many of the predicted metabolic properties of w Mel, such as the focus on amino acid transport and the presence of limited carbohydrate metabolism, are similar to those found in Rickettsia. A major difference with the Rickettsia spp. is the absence of the ADP–ATP exchanger protein in w Mel. In Rickettsia this protein is used to import ATP from the host, thus allowing these species to be direct energy scavengers . This likely explains the presence of glycolysis in w Mel but not Rickettsia. An inability to obtain ATP from its host also helps explain the presence of pathways for the synthesis of the purines AMP, IMP, XMP, and GMP in w Mel but not Rickettsia. Other pathways present in w Mel but not Rickettsia include threonine degradation (described above), riboflavin biosynthesis, pyrimidine metabolism (i.e., from PRPP to UMP), and chelated iron uptake (using a single ABC transporter). The two Rickettsia species have a relatively large complement of predicted transporters for osmoprotectants, such as proline and glycine betaine, whereas w Mel possesses only two of these systems. | 15024419_p23 | 15024419 | Metabolism and Transport | 4.452089 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
The w Mel genome is predicted to encode few proteins for regulatory responses. Three genes encoding two-component system subunits are present: two sensor histidine kinases and one response regulator . Only six strong candidates for transcription regulators were identified: a homolog of arginine repressors , two members of the TenA family of transcription activator proteins , a homolog of ctrA , a transcription regulator for two component systems in other α-Proteobacteria , and two σ factors . There are also seven members of one paralogous family of proteins that are distantly related to phage repressors (see above), although if they have any role in transcription, it is likely only for phage genes. Such a limited repertoire of regulatory systems has also been reported in other endosymbionts and has been explained by the apparent highly predictable and stable environment in which these species live . | 15024419_p24 | 15024419 | Regulatory Responses | 4.322395 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
The mechanisms by which Wolbachia infect host cells and by which they cause the diverse phenotypic effects on host reproduction and fitness are poorly understood, and the w Mel genome helps identify potential contributing factors. A complete Type IV secretion system, portions of which have been reported in earlier studies, is present. The complete genome sequence shows that in addition to the five vir genes previously described from Wolbachia w KueYO , an additional four are present in w Mel. Of the nine w Mel vir ORFs, eight are arranged into two separate operons. Similar to the single operon identified in w Tai and w KueYO, the w Mel virB8 , virB9 , virB10 , virB11 , and virD4 CDSs are adjacent to wspB , forming a 7 kb operon . The second operon contains virB3 , virB4 , and virB6 as well as four additional non- vir CDSs, including three putative membrane-spanning proteins, that form part of a 15.7 kb operon . Examination of the Rickettsia conorii genome shows a similar orga-nization . The observed conserved gene order for these genes between these two genomes suggests that the putative membrane-spanning proteins could form a novel and, possibly, integral part of a functioning Type IV secretion system within these bacteria. Moreover, reverse transcription (RT)-PCRs have confirmed that wspB and WD0853–WD0856 are each expressed as part of the two vir operons and further indicate that these additional encoded proteins are novel components of the Wolbachia Type IV secretion system . | 15024419_p25 | 15024419 | Host–Symbiont Interactions | 4.510443 | biomedical | Study | [
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] | [
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] | en | 0.999996 |
In addition to the two major vir clusters, a paralog of virB8 is also present in the w Mel genome. WD0818 is quite divergent from virB8 and, as such, does not appear to have resulted from a recent gene duplication event. RT-PCR experiments have failed to show expression of this CDS in w Mel-infected Drosophila (data not shown). PCR primers were designed to all CDSs of the w Mel Type IV secretion system and used to successfully amplify orthologs from the divergent Wolbachia strains w Ri and w AlbB (data not shown). We were able to detect orthologs to all of the w Mel Type IV secretion system components as well as most of the adjacent non- vir CDSs, suggesting that this system is conserved across a range of A- and B-group Wolbachia . An increasing body of evidence has highlighted the importance of Type IV secretion systems for the successful infection, invasion, and persistence of intracellular bacteria within their hosts . It is likely that the Type IV system in Wolbachia plays a role in the establishment and maintenance of infection and possibly in the generation of reproductive phenotypes. | 15024419_p26 | 15024419 | Host–Symbiont Interactions | 4.367495 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
Genes involved in pathogenicity in bacteria have been found to be frequently associated with regions of anomalous nucleotide composition, possibly owing to transfer from other species or insertion into the genome from plasmids or phage. In the four such regions in w Mel (see above; see Table 3 ), some additional candidates for pathogenicity-related activities are present including a putative penicillin-binding protein , genes predicted to be involved in cell wall synthesis and a multidrug resistance protein . In addition, we have identified a cluster of genes in one of the phage regions that may also have some role in host–symbiont interactions. This cluster is embedded within the WO-B phage region of the genome and contains many genes that encode proteins with putative roles in the synthesis and degradation of surface polysaccharides, including a UDP-glucose 6-dehydrogenase . Since this cluster appears to be normal in terms of phylogeny relative to other genes in the genome (i.e., the genes in this region have normal w Mel nucleotide composition and branch in phylogenetic trees with genes from other α-Proteobacteria), it is not likely to have been acquired from other species. However, it is possible that these genes can be transferred among Wolbachia strains via the phage, which in turn could lead to some variation in host–symbiont interactions between Wolbachia strains. | 15024419_p27 | 15024419 | Host–Symbiont Interactions | 4.41354 | biomedical | Study | [
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] | [
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] | en | 0.999995 |
Of particular interest for host-interaction functions are the large number of genes that encode proteins that contain ankyrin repeats ( Table 4 ). Ankyrin repeats, a tandem motif of around 33 amino acids, are found mainly in eukaryotic proteins, where they are known to mediate protein–protein interactions . While they have been found in bacteria before, they are usually present in only a few copies per species. w Mel has 23 ankyrin repeat-containing genes, the most currently described for a prokaryote, with C. burnetti being next with 13. This is particularly striking given w Mel's relatively small genome size. The functions of the ankyrin repeat-containing proteins in w Mel are difficult to predict since most have no sequence similarity outside the ankyrin domains to any proteins of known function. Many lines of evidence suggest that the w Mel ankyrin domain proteins are involved in regulating host cell-cycle or cell division or interacting with the host cytoskeleton: (i) many ankyrin-containing proteins in eukaryotes are thought to be involved in linking membrane proteins to the cytoskeleton ; (ii) an ankyrin-repeat protein of Ehrlichia phagocytophila binds condensed chromatin of host cells and may be involved in host cell-cycle regulation ; (iii) some of the proteins that modify the activity of cell-cycle-regulating proteins in D. melanogaster contain ankyrin repeats ; and (iv) the Wolbachia strain that infects the wasp Nasonia vitripennis induces cytoplasmic incompatibility, likely by interacting with these same cell-cycle proteins . Of the ankyrin-containing proteins in w Mel, those worth exploring in more detail include the several that are predicted to be surface targeted or secreted ( Table 4 ) and thus could be targeted to the host nucleus. It is also possible that some of the other ankyrin-containing proteins are secreted via the Type IV secretion system in a targeting signal independent pathway. We call particular attention to three of the ankyrin-containing proteins , which are among the very few genes, other than those encoding components of the translation apparatus, that have significantly biased codon usage relative to what is expected based on GC content, suggesting they may be highly expressed. | 15024419_p28 | 15024419 | Host–Symbiont Interactions | 4.461173 | biomedical | Study | [
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] | [
0.9990837574005127,
0.0003020532603841275,
0.0005129873752593994,
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] | en | 0.999995 |
Analysis of the w Mel genome reveals that it is unique among sequenced genomes of intracellular organisms in that it is both streamlined and massively infected with mobile genetic elements. The persistence of these elements in the genome for apparently long periods of time suggests that w Mel is inefficient at getting rid of them, likely a result of experiencing severe population bottlenecks during every cycle of transovarial transmission as well as during sweeps through host populations. Integration of evolutionary reconstructions and genome analysis (phylogenomics) has provided insights into the biology of Wolbachia , helped identify genes that likely play roles in the unusual effects Wolbachia have on their host, and revealed many new details about the evolution of Wolbachia and mitochondria. Perhaps most importantly, future studies of Wolbachia will benefit both from this genome sequence and from the ability to study host–symbiont interactions in a host ( D. melanogaster ) well-suited for experimental studies. | 15024419_p29 | 15024419 | Conclusions | 4.339721 | biomedical | Study | [
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] | en | 0.999995 |
w Mel DNA was obtained from D. melanogaster yw 67c23 flies that naturally carry the w Mel infection. w Mel was purified from young adult flies on pulsed-field gels as described previously . Plugs were digested with the restriction enzyme AscI (GG^CGCGCC), which cuts the bacterial chromosome twice , aiding in the entry of the DNA into agarose gels. After electrophoresis, the resulting two bands were recovered from the gel and stored in 0.5 M EDTA (pH 8.0). DNA was extracted from the gel slices by first washing in TE (Tris–HCl and EDTA) buffer six times for 30 min each to dilute EDTA followed by two 1-h washes in β-agarase buffer (New England Biolabs, Beverly, Massachusetts, United States). Buffer was then removed and the blocks melted at 70°C for 7 min. The molten agarose was cooled to 40°C and then incubated in β-agarase (1 U/100 μl of molten agarose) for 1 h. The digest was cooled to 4°C for 1 h and then centrifuged at 4,100 × g max for 30 min at 4°C to remove undigested agarose. The supernatant was concentrated on a Centricon YM-100 microconcentrator (Millipore, Bedford, Massachusetts, United States) after prerinsing with 70% ethanol followed by TE buffer and, after concentration, rinsed with TE. The retentate was incubated with proteinase K at 56°C for 2 h and then stored at 4°C. w Mel DNA for gap closure was prepared from approximately 1,000 Drosophila adults using the Holmes–Bonner urea/phenol:chloroform protocol to prepare total fly DNA. | 15024419_p30 | 15024419 | Purification/source of DNA | 4.27898 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
The complete genome sequence was determined using the whole-genome shotgun method . For the random shotgun-sequencing phase, libraries of average size 1.5–2.0 kb and 4.0–8.0 kb were used. After assembly using the TIGR Assembler , there were 78 contigs greater than 5000 bp, 186 contigs greater than 3000 bp, and 373 contigs greater than 1500 bp. This number of contigs was unusually high for a 1.27 Mb genome. An initial screen using BLASTN searches against the nonredundant database in GenBank and the Berkeley Drosophila Genome Project site ( http://www.fruitfly.org/blast/ ) showed that 3,912 of the 10,642 contigs were likely contaminants from the Drosophila genome. To aid in closure, the assemblies were rerun with all sequences of likely host origin excluded. Closure, which was made very difficult by the presence of a large amount of repetitive DNA (see below), was done using a mix of primer walking, generation, and sequencing of transposon-tagged libraries of large insert clones and multiplex PCR . The final sequence showed little evidence for polymorphism within the population of Wolbachia DNA. In addition, to obtain sequence across the AscI-cut sites, PCR was performed on undigested DNA. It is important to point out that the reason significant host contamination does not significantly affect symbiont genome assembly is that most of the Drosophila contigs were small due to the approximately 100-fold difference in genome sizes between host (approximately 180 Mb) and w Mel (1.2 Mb). | 15024419_p31 | 15024419 | Library construction/sequencing/closure | 4.291992 | biomedical | Study | [
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] | [
0.9993413090705872,
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0.0002737128525041044,
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] | en | 0.999996 |
Since it has been suggested that Wolbachia and their hosts may undergo lateral gene transfer events , genome assemblies were rerun using all of the shotgun and closure reads without excluding any sequences that appeared to be of host origin. Only five assemblies were found to match both the D. melanogaster genome and the w Mel assembly. Primers were designed to match these assemblies and PCR attempted from total DNA of w Mel infected D. melanogaster . In each case, PCR was unsuccessful, and we therefore presume that these assemblies are the result of chimeric cloning artifacts. The complete sequence has been given GenBank accession ID AE017196 and is available at http://www.tigr.org/tdb . | 15024419_p32 | 15024419 | Library construction/sequencing/closure | 4.145427 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
Repeats were identified using RepeatFinder , which makes use of the REPuter algorithm to find maximal-length repeats. Some manual curation and BLASTN and BLASTX searches were used to divide repeat families into different classes. | 15024419_p33 | 15024419 | Repeats | 3.295487 | biomedical | Study | [
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] | [
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] | en | 0.999996 |
Identification of putative protein-encoding genes and annotation of the genome was done as described previously . An initial set of ORFs likely to encode proteins (CDS) was identified with GLIMMER . Putative proteins encoded by the CDS were examined to identify frameshifts or premature stop codons compared to other species. The sequence traces for each were reexamined and, for some, new sequences were generated. Those for which the frameshift or premature stops were of high quality were annotated as “authentic” mutations. Functional assignment, identification of membrane-spanning domains, determination of paralogous gene families, and identification of regions of unusual nucleotide composition were performed as described previously . Phylogenomic analysis was used to aid in functional predictions. Alignments and phylogenetic trees were generated as described . | 15024419_p34 | 15024419 | Annotation | 4.196932 | biomedical | Study | [
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] | [
0.99921715259552,
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] | en | 0.999997 |
All putative w Mel proteins were searched using BLASTP against the predicted proteomes of published complete organismal genomes and a set of complete plastid, mitochondrial, plasmid, and viral genomes. The results of these searches were used (i) to analyze the phylogenetic profile , (ii) to identify putative lineage-specific duplications (those proteins with a top E -value score to another protein from w Mel), and (iii) to determine the presence of homologs in different species. Orthologs between the w Mel genome and that of the two Rickettsia species were identified by requiring mutual best-hit relationships among all possible pairwise BLASTP comparisons, with some manual correction. Those genes present in both Rickettsia genomes as well as other bacterial species, but not w Mel, were considered to have been lost in the w Mel branch (see Table S3 ). Genes present in only one or two of the three species were considered candidates for gene loss or lateral transfer and were also used to identify possible biological differences between these species (see Table S3 ). For the w Mel genes not in the Rickettsia genomes, proteins were searched with BLASTP against the TIGR NRAA database. Protein sequences of their homologs were aligned with CLUSTALW and manually curated. Neighbor-joining trees were constructed using the PHYLIP package. | 15024419_p35 | 15024419 | Comparative genomics | 4.204727 | biomedical | Study | [
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] | [
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0.00008603958849562332
] | en | 0.999996 |
For phylogenetic analysis, the set of all 38 proteins encoded in both the Marchantia polymorpha and Reclinomonas americana mitochondrial genomes were collected. Acanthamoeba castellanii was excluded due to high divergence and extremely long evolutionary branches. Six genes were excluded from further analysis because they were too poorly conserved for alignment and phylogenetic analysis ( nad7 , rps10 , sdh3 , sdh4 , tatC , and yejV ), leaving 32 genes for investigation: atp6 , atp9 , atpA , cob , cox1 , cox2 , cox3 , nad1 , nad2 , nad3 , nad4 , nad4L , nad5 , nad6 , nad9 , rpl16 , rpl2 , rpl5 , rpl6 , rps1 , rps11 , rps12 , rps13 , rps14 , rps19 , rps2 , rps3 , rps4 , rps7 , rps8 , yejR , and yejU . Using FASTA with the mitochondrial proteins as a query, homologs were identified from the genomes of seven α-Proteobacteria: two intracellular symbionts ( W. pipientis w Mel and Rickettsia prowazekii ) and five free-living forms ( Sinorhozobium loti , Agrobacterium tumefaciens , Brucella melitensis , Mesorhizobium loti , and Rhodopseudomonas sp.). Escherichia coli and Neisseria meningitidis were used as outgroups. Caulobacter crescentus was excluded from analysis because homologs of some of the 32 genes were not found in the current annotation. In the event that more than one homolog was identified per genome, the one with the greatest sequence identity to the mitochondrial query was retrieved. Proteins were aligned using CLUSTALW and concatenated. To reduce the influence of poorly aligned regions, all sites that contained a gap at any position were excluded from analysis, leaving 6,776 positions per genome for analysis. The data contained extreme amino acid bias: all sequences failed the χ 2 test at p = 0.95 for deviation from amino acid frequency distribution assumed under either the JTT or mtREV24 models as determined with PUZZLE . When the data were iteratively purged of highly variable sites using the method described , amino acid composition gradually came into better agreement with acid frequency distribution assumed by the model. The longest dataset in which all sequences passed the χ 2 test at p = 0.95 consisted of the 3,100 least polymorphic sites. PROTML analyses of the 3,100-site data using the JTT model detected mitochondria as sisters of the five free-living α-Proteobacteria with low (72%) support, whereas PUZZLE, using the same data, detected mitochondria as sisters of the two intracellular symbionts, also with low (85%) support. This suggested the presence of conflicting signal in the less-biased subset of the data. Therefore, protein log determinants (LogDet) were used to infer distances from the 6,776-site data, since the method can correct for amino acid bias , and Neighbor-Net was used to display the resulting matrix, because it can detect and display conflicting signal. The result shows both signals. In no analysis was a sister relationship between Rickettsia and mitochondria detected. | 15024419_p36 | 15024419 | Phylogenetic analysis of mitochondrial proteins | 4.380245 | biomedical | Study | [
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] | [
0.9991827607154846,
0.00024591886904090643,
0.0004731059889309108,
0.00009820738341659307
] | en | 0.999998 |
For analyses of individual genes, the 63 proteins encoded in the Reclinomonas mitochondrial genome were compared with FASTA to the proteins from 49 sequenced eubacterial genomes, which included the α-Proteobacteria shown in Figure 5 , R. conorii , and Magnetococcus MC1, one of the more divergent α-Proteobacteria. Of those proteins, 50 had sufficiently well-conserved homologs to perform phylogenetic analyses. Homologs were aligned and subjected to phylogenetic analysis with PROTML . | 15024419_p37 | 15024419 | Phylogenetic analysis of mitochondrial proteins | 4.111969 | biomedical | Study | [
0.9994385838508606,
0.0002531270729377866,
0.00030824943678453565
] | [
0.999525785446167,
0.00021152755653019994,
0.00020959558605682105,
0.00005313090514391661
] | en | 0.999997 |
To compare wspB sequences from different Wolbachia strains, PCR was done on total DNA extracted from the following sources: w Ri was obtained from infected adult D. simulans , Riverside strain; w AlbB was obtained from the infected Aa23 cell line , and D. immitis Wolbachia was extracted from adult worm tissue. DNA extraction and PCR were done as previously described with wspB -specific primers ( wspB -F, 5′-TTTGCAAGTGAAACAGAAGG and wspB -R, 5′-GCTTTGCTGGCAAAATGG). PCR products were cloned into pGem-T vector (Promega, Madison, Wisconsin, United States) as previously described and sequenced . These sequences were compared to previously sequenced wsp genes for the same Wolbachia strains . The four partial wsp sequences were aligned using CLUSTALV based on the amino acid translation of each gene and similarly with the wspB sequences. Genetic distances were calculated using the Kimura 2 parameter method and are reported in Table S5 . | 15024419_p38 | 15024419 | Analysis of wspB sequences | 4.127318 | biomedical | Study | [
0.9995143413543701,
0.00024871420464478433,
0.0002369689755141735
] | [
0.9995152950286865,
0.00017518983804620802,
0.00025228658341802657,
0.00005732616045861505
] | en | 0.999997 |
To determine whether the vir -like CDSs, as well as adjacent ORFs, were actively expressed within w Mel as two polycistronic operons, RT-PCR was used. Total RNA was isolated from infected D. melanogaster yw 67c23 adults using Trizol reagent (Invitrogen, Carlsbad, California, United States) and cDNA synthesized using SuperScript III RT (Invitrogen) using primers wspB R, WD0817R, WD0853R, and WD0852R. RNA isolation and RT were done according to manufacturer's protocols, with the exception that suggested initial incubation of RNA template and primers at 65°C for 5 min and final heat denaturation of RT-enzyme at 70°C for 15 min were not done. PCR was done using r Taq (Takara, Kyoto, Japan), and several primer sets were used to amplify regions spanning adjacent CDSs for most of the two operons. For operon virB3-WD0853, the following primers were used: ( virB3 - virB4 )F, ( virB3 - virB4 )R, F, R, F, R, F, R. For operon virB8 - wspB , the following primers were used: ( virB8 - virB9 )F, ( virB8 - virB9 )R, ( virB9 - virB11 )F, ( virB9 - virB11 )R, ( virB11 - virD4 )F, ( virB11 - virD4 )R, ( virD4 - wspB )F, and ( virD4 - wspB )R. The coexpression of virB4 and virB6 , as well as WD0855 and WD0854, was confirmed within the putative virB3 -WD0853 operon using nested PCR with the following primers: ( virB4 - virB6 )F1, ( virB4 - virB6 )R1, ( virB4 - virB6 )F2, ( virB4 - virB6 )R2, F1, R1, F2, and R2. All ORFs within the putative virB8 - wspB operon were shown to be coexpressed and are thus considered to be a genuine operon. All products were amplified only from RT-positive reactions . Primer sequences are given in Table S9 . | 15024419_p39 | 15024419 | Type IV secretion system | 4.25507 | biomedical | Study | [
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0.0003503205953165889,
0.0002718681644182652
] | [
0.9993599057197571,
0.0003052329702768475,
0.0002547305775806308,
0.0000801053101895377
] | en | 0.999995 |
The complete sequence for w Mel has been given GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession ID number AE017196 and is available through the TIGR Comprehensive Microbial Resourceat http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=dmg | 15024419_p40 | 15024419 | Accession Numbers | 1.716569 | biomedical | Other | [
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] | en | 0.999997 |
The GenBank accession numbers for other sequences discussed in this paper are AF020059 ( Wolbachia sp. w AlbB outer surface protein precursor wsp gene), AF020070 ( Wolbachia sp. w Ri outer surface protein precursor wsp gene), AJ252062 ( Wolbachia endosymbiont of D. immitis sp. gene for surface protein), AJ580921 ( Wolbachia endosymbiont of D. immitis partial wspB gene for Wolbachia surface protein B), AJ580922 ( Wolbachia endosymbiont of A. albopictus partial wspB gene for Wolbachia surface protein B), and AJ580923 ( Wolbachia endosymbiont of D. simulans partial wspB gene for Wolbachia surface protein B). | 15024419_p41 | 15024419 | Accession Numbers | 2.952609 | biomedical | Other | [
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] | [
0.15327952802181244,
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0.00147471041418612
] | en | 0.999996 |
The number and type of genetic changes that control morphological and physiological changes during vertebrate evolution are not yet known. The evolutionary history of threespine sticklebacks (Gasterosteus aculeatus) provides an unusual opportunity to directly study the genetic architecture of adaptive divergence in natural populations. At the end of the last ice age, marine sticklebacks colonized newly formed freshwater environments throughout the Northern Hemisphere. Over the last 10,000 to 15,000 years, these fish have adapted to a wide range of new ecological conditions, giving rise to diverse populations with striking differences in morphology, physiology, and behavior . Major changes in the bony armor have evolved repeatedly in different locations, and several hypotheses have been proposed to explain this morphological transformation, including response to changes in calcium availability , stream gradients , or temperature, salinity, or other factors that may vary in parallel with climate ; or exposure to different types of predators . | 15069472_p0 | 15069472 | Introduction | 4.225135 | biomedical | Study | [
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Three distinctive patterns of body armor, now known as the “lateral plate morphs,” have been recognized as one of the most distinguishing characteristics in sticklebacks since at least the early 1800s . Most marine sticklebacks have a continuous row of bony plates that covers the lateral side of the body from head to tail . In contrast, many freshwater sticklebacks show substantial reductions in total plate number, developing either as “partial morphs,” which lose plates in the middle of the row (not shown), or as “low morphs,” which retain only a few plates at the anterior end . The anterior plates present in low morphs are the first to form during larval development. In contrast, the middle plates absent in partial morphs are the last to form during normal development . Thus, the adult plate patterns of low and partial morphs resemble early developmental stages of plate patterns in complete morphs, and paedomorphosis has been proposed as a possible explanation for the repeated evolution of low and partial morphs from completely plated ancestors . | 15069472_p1 | 15069472 | Introduction | 4.300195 | biomedical | Study | [
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This dramatic variation in lateral plate patterning has led to repeated efforts to determine the genetic basis of the major plate morphs. Previous studies have shown that plate morphs are reproducibly inherited in the laboratory and that crosses between different morphs generate relatively simple ratios of the three major phenotypes among the progeny. Based on these qualitative results, at least six different genetic models have been proposed for lateral plate patterns in sticklebacks. The simplest models proposed a single major locus with alternative alleles (A and a) . The A allele was first proposed to be incompletely dominant to the a allele, generating either complete (AA), partial (Aa), or low-plated (aa) fish . In other populations, the A allele may be completely dominant to the a allele, producing either complete (AA, Aa) or low-plated (aa) fish, but no partials . More complicated models have proposed two major loci controlling plate inheritance (with alternative alleles A, a and B, b ). In one of these models, both major loci contribute equally to plate phenotype, and the total number of A and B alleles determines whether fish develop as complete (three or more A or B alleles), partial (two A or B alleles), or low-plated fish (one or less A or B allele) . Additional models have proposed either epistatic interactions between a single major locus and one modifier locus, or the presence of more than two alternative alleles at the major locus to account for variant results in some populations . All of these models were proposed before the development of genomewide genetic markers for sticklebacks and have never been tested by linkage mapping. In this study, we take advantage of these recently developed tools to examine the genetic basis of variation in lateral plate phenotypes in natural populations of sticklebacks. | 15069472_p2 | 15069472 | Introduction | 4.370673 | biomedical | Study | [
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To directly analyze the number and location of genetic loci that control plate phenotypes, we crossed a completely plated marine fish with a low-plated benthic fish from Paxton Lake, British Columbia. Three hundred sixty progeny from a single F2 family (Cross 1) were examined in detail for the pattern, number, and size of lateral plates and then genotyped for the inheritance of different alleles at 160 polymorphic loci distributed across all linkage groups. The segregation of plate phenotypes was compared to the segregation of all genetic markers using quantitative trait loci (QTL) analysis . Significance thresholds for detecting linkage were chosen using conservative criteria for genomewide linkage mapping in noninbred populations . | 15069472_p3 | 15069472 | Results | 4.144583 | biomedical | Study | [
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] | [
0.9995840191841125,
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When plate morph was scored as a qualitative trait, a highly significant QTL on linkage group (LG) 4 was detected . The genotype of the QTL on LG 4 was highly predictive of the major plate morph that developed in a fish. Almost all fish that carried two alleles from the complete morph grandparent in the LG 4 region (hereafter referred to as “AA” animals) showed the complete pattern, whereas fish that carried two alleles from the low morph grandparent in this region (hereafter referred to as “aa” animals) showed the low pattern. In contrast, most fish with one allele from the complete grandparent and one allele from the low grandparent (hereafter referred to as “Aa” animals) developed as either complete or partial fish . | 15069472_p4 | 15069472 | Results | 4.085275 | biomedical | Study | [
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0.9993732571601868,
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] | en | 0.999997 |
When total plate number was scored, the same major LG 4 chromosome region accounted for more than 75% of the total variance in plate number of F2 fish. Three additional QTL were detected that had significant effects on plate number in Aa animals . Increasing the number of benthic alleles at any of the individual modifiers led to a reduction in mean total plate number, even in the heterozygous state ( Table 1 ). Increasing the number of benthic alleles at the three modifiers considered together caused a more than 2-fold reduction in mean plate number of Aa animals, largely accounting for whether Aa fish developed as either complete, partial, or low morphs . Increasing the number of benthic alleles at the same modifier loci also led to a 2-fold reduction in the mean plate number of aa animals but had relatively little effect on the plate number of AA animals . Taken together, these results suggest that at least four different loci influence lateral plate phenotypes in this cross. Homozygosity at the major locus largely determines whether fish develop as low (aa) or complete (AA) morphs, while the modifier loci affect the actual number of plates, particularly in Aa and aa animals. | 15069472_p5 | 15069472 | Results | 4.200022 | biomedical | Study | [
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] | [
0.9995465874671936,
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The size of individual lateral plates varies significantly between different stickleback populations . Although this trait has not been systematically analyzed in previous stickleback crosses, studies of meristic characters in other vertebrates suggest that the size and number of repeating skeletal elements can be controlled separately . When height and width of specific plates were analyzed, we detected three QTL that accounted for a significant percentage of plate size variability in the cross . Increasing the number of benthic alleles at these loci led to a progressive reduction in plate size . Two of the three plate size QTL mapped to the same chromosome regions that also affected plate morph or plate number, suggesting that the pattern, number, and size of plates may be controlled by the same or linked genes on LGs 4 and 7 . In contrast, the QTL affecting lateral plate size mapped to different locations than most QTL controlling the size of dorsal spine and pelvic structures , suggesting that the size of different bones are controlled separately in the stickleback skeleton. | 15069472_p6 | 15069472 | Results | 4.196465 | biomedical | Study | [
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] | [
0.9995480179786682,
0.00018666287360247225,
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Some of the differences in previously published models of stickleback plate genetics could be due to different genetic mechanisms operating in different populations. To compare the genetic architecture of armor plate patterning in a separate population located over 1300 km from Paxton Lake, we crossed fish from an unusual stickleback population in Friant, California, which is largely dimorphic for complete and low fish with very few partials. A cross between a Friant complete and a Friant low-plated fish resulted in nearly equal numbers of complete and low progeny , consistent with previous crosses from this population . Genotyping studies with microsatellite markers linked to the major and minor QTL defined above showed very tight concordance between lateral plate phenotype and genotype near the same major locus on LG 4 that was seen in Cross 1 (LOD = 11.1). All fish with an inferred Aa genotype at the major locus on LG 4 were completely plated in this cross, suggesting that Aa fish develop more plates in Cross 2 than in Cross 1. This could be due to differences in the dominance relationship of the particular alleles at the LG 4 locus in the Friant population , or to modification of dominance by the different genetic backgrounds in the two crosses. Although the number of animals in Cross 2 was small, significant differences in the mean total plate count of low fish could also be detected in animals that inherited different alleles at microsatellites linked to two of the modifier QTL detected in Cross 1 . Overall, these results suggest that both plate morph and plate number are controlled by similar chromosome regions in different populations. | 15069472_p7 | 15069472 | Results | 4.25595 | biomedical | Study | [
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] | [
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To further test whether the same major locus on LG 4 controls armor plate reduction in both populations, we carried out genetic complementation crosses between two low female fish from Friant and one low male fish from Paxton Lake. All 84 progeny developed as low morphs, suggesting that the low-plated phenotype in both populations is likely to be due to the same major locus on LG 4. | 15069472_p8 | 15069472 | Results | 4.014111 | biomedical | Study | [
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] | [
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This study reports the first genomewide linkage mapping of lateral plate phenotypes in crosses between major stickleback plate morphs. Our results confirm previous suggestions that dramatic changes in lateral plate patterning can be controlled by one locus of major effect . This major locus on LG 4 can cause a greater than 5-fold change in total plate number and is sufficient to switch the overall morphology of a fish between the complete, partial, and low-plated states. The dramatic phenotypic effects of this locus likely explain why three types of sticklebacks have long been recognized in natural populations . Further molecular studies will be required to determine whether there are one or multiple mutations in the LG 4 region that account for the major QTL. | 15069472_p9 | 15069472 | QTL Architecture | 4.213182 | biomedical | Study | [
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Plate number within the complete, partial, and low morphs also varies between fish from different locations. Previous studies suggest that sticklebacks with small changes in plate number show differential survival when exposed to predators, suggesting that selection may fine tune the exact number of plates in different environments . We have identified three modifier QTL that cause changes in plate number within all morphs but are unlinked to the major locus. The individual phenotypic effects of these QTL can be as small as a single plate per side ( Table 1 ), while the combined mean effects of the QTL can be as large as 15 plates per side . The number of modifier QTL is larger than predicted in previous models. We suspect that this is because of the general difficulty of predicting genetic architecture from simple phenotypic ratios of progeny in crosses that are segregating more than one or two genes. The magnitude of the phenotypic effects of the modifiers, their linkage relationships, and interactions with the major locus could not be predicted accurately from previous studies, highlighting the value of genomewide linkage mapping for studying the genetic architecture of major morphological variation in natural populations. | 15069472_p10 | 15069472 | QTL Architecture | 4.202206 | biomedical | Study | [
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] | [
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] | en | 0.999995 |
Postglacial freshwater stickleback populations are thought to be derived from completely plated marine ancestors . At all of the plate QTL detected in Cross 1, the net effect of the freshwater alleles from the Paxton benthic grandparent is to cause a progressive reduction in the size or number of armor plates ( Table 1 ). All of the QTL that affect plate morph or plate number also have significant effects in the heterozygous state, showing that plate reduction is likely to evolve through semiadditive genetic changes, rather than through purely recessive or purely dominant mutations. Theoretical studies suggest that semiadditive mutations can be fixed more quickly than purely recessive or dominant mutations when they begin at low frequency, although the overall probability of fixation also depends on whether the mutations arise de novo or are originally present in a founder population . Strong selection on a small number of chromosome regions that have large, semiadditive effects may help explain how dramatic changes in lateral plate patterns have evolved relatively quickly in postglacial stickleback populations. | 15069472_p11 | 15069472 | QTL Architecture | 4.196543 | biomedical | Study | [
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] | [
0.9994832277297974,
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] | en | 0.999999 |
Our mapping and complementation results suggest that the same major locus on LG 4 causes major changes in plate pattern in both the Paxton benthic and Friant populations. Phenotypic reduction of lateral plates almost certainly evolved separately in these different locations, given the geographic distance between them , the presence of completely plated fish in the marine environment separating the sites, and previous studies showing that sticklebacks in nearby lakes have independent mitochondrial haplotypes . Additional complementation crosses between low-plated fish from Friant and other California populations , Paxton benthic fish and pelvic-reduced fish from Iceland , and low-plated populations from British Columbia and Japan also produce low-plated progeny. Thus genetic changes at the same major locus may underlie low-plated phenotypes at numerous locations around the world. | 15069472_p12 | 15069472 | Parallel Evolution | 4.203407 | biomedical | Study | [
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The present study provides the first genetic mapping evidence that some of the chromosome regions controlling smaller quantitative variation in plate number may also be used repeatedly in different populations. The QTL on LG 26 in Cross 1 maps to a similar position as a QTL influencing plate number within low morph fish from Priest Lake, British Columbia . This QTL is also associated with significant variation in plate number of low morphs of the Friant population (Cross 2), suggesting that this chromosomal region on LG 26 contributes to plate number variation in at least three different populations: Paxton, Priest, and Friant sticklebacks. | 15069472_p13 | 15069472 | Parallel Evolution | 4.144471 | biomedical | Study | [
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Recent studies suggest that the same genes are also used repeatedly when pigmentation and larval cuticle phenotypes have evolved in parallel in different fly populations or when melanism has evolved independently in birds and mammals . Repeated use of particular genes may thus be a common theme in parallel evolution of major morphological changes in natural populations of both invertebrates and vertebrates. | 15069472_p14 | 15069472 | Parallel Evolution | 3.917622 | biomedical | Study | [
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] | [
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Why might some genes be used preferentially when similar phenotypes evolve in parallel in wild populations? Alleles that cause plate reduction may already be present at low frequency in marine populations. In that case, parallel phenotypic evolution could occur by repeated selection for the same preexisting alleles in different freshwater locations. Alternatively, some genes may be particularly susceptible to de novo mutations, either because of the size or structure of coding and regulatory regions, or the presence of hotspots for recombination, insertion, or deletion. Finally, only a limited number of either old or new mutations may actually be capable of producing a specific phenotype without also causing deleterious effects on fitness. Mutations with the largest positive selection coefficients will be fixed most rapidly in evolving populations, and this may lead to parallel selection for mutations in the same genes in different populations. | 15069472_p15 | 15069472 | Parallel Evolution | 4.238314 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
A major goal for future work will be to identify the actual genes and mutations that cause parallel evolution of adaptive traits in wild sticklebacks. This study identifies specific markers that are closely linked to chromosome regions that control the pattern, number, and size of lateral plates. With the recent development of BAC libraries and physical maps of the stickleback genome, it should be possible to use forward genetic approaches to identify the genes responsible for the repeated evolution of major morphological transformations in stickleback armor . Cloning and sequencing of such genes will make it possible to determine the molecular mechanisms that underlie parallel evolution in natural populations and should provide new insight into the nature of genetic, genomic, developmental, and ecological constraints that operate as new characteristics appear during the adaptive evolution of vertebrates. | 15069472_p16 | 15069472 | Parallel Evolution | 4.180039 | biomedical | Study | [
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] | [
0.999226450920105,
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For Cross 1, a wild-caught, completely plated marine female from Onnechikappu stream on the east coast of Hokkaido Island, Japan, was crossed to a wild-caught, low-plated benthic male from Paxton Lake, British Columbia. Both parents showed morphologies typical of the marine and benthic populations at their respective collecting sites. The specific populations were chosen because the large average body size of both parents and the estimated divergence between eastern and western Pacific Ocean fish were expected to help maximize the size of the progeny, the number of offspring per clutch, and the informativeness of microsatellites and other markers for genetic mapping. F1 progeny were raised to maturity in 30-gallon aquaria and were mated in pairs. Approximately 2600 F2 progeny were raised to a standard length of greater than 28 mm under the same conditions (30-gallon aquaria in a single 18°C room with 16 hours of light and eight hours of dark per day and twice daily feeding of brine shrimp or frozen blood worms). Although limited phenotypic plasticity has been reported for development of some trophic characters in sticklebacks , previous studies have shown that differences in plate number of wild-caught sticklebacks are stable and reproducible when fish are raised under laboratory conditions . A total of 360 full siblings from a single F2 family were used for genotypic and phenotypic analysis in this study. For Cross 2, one wild-caught, completely plated female from Friant, California, was crossed to one wild-caught, low-plated male from Friant, California. A total of 58 F1 progeny were raised to a standard length of greater than 28 mm in a ZMOD (Marine Biotech, Beverly, Massachusetts, United States). For the complementation cross, two wild-caught, low-plated females from Friant were crossed to one wild-caught, low-plated benthic male from Paxton Lake, British Columbia. At total of 84 F1 progeny were raised to a standard length of greater than 28 mm in 30-gallon aquaria. | 15069472_p17 | 15069472 | Fish crosses and husbandry | 4.189758 | biomedical | Study | [
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Genotyping of microsatellite markers was performed and analyzed essentially as described in Peichel et al. . Some PCR products were analyzed on a 48-capillary array on an ABI3730xl with GeneMapper v3.0 software and GeneScan 500 LIZ (Applied Biosystems, Foster City, California, United States) used as an internal size standard. A total of 160 markers were analyzed in Cross 1, including 144 previously described microsatellite markers , the genes Pitx1 , Pitx2 (Stn220), and Tbx4 (Stn221), and 13 new markers: Bmp6 gene and 12 additional microsatellites (Stn210–219, 222–223). A polymorphism within the 3′ UTR of the Bmp6 gene was genotyped using single strand conformation polymorphism analysis with MDE Gel Solution (BioWhittaker Molecular Applications, Rockland, Maine, United States). PCR bands were visualized using autoradiography. PCR conditions were the same as for the microsatellite markers except 2.5 mM MgCl 2 and 10% DMSO were used. Primers for Bmp6 genotyping are: Bmp6F1: 5′ CCCGGTTT AA ATCCTCATCC and Bmp6R1: 5′ AGGAGGTGATTGACAGCTCG. | 15069472_p18 | 15069472 | Genotyping | 4.158587 | biomedical | Study | [
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Fish were stained with alizarin red to detect skeletal structures as described in Peichel et al. . Lateral plates were counted on both sides of each fish. For QTL mapping, the total plate number of both sides was used. Plate width was measured on the first lateral plate located under the first dorsal spine and above the ascending process of the pelvis. Plate height was measured on the lateral plate posterior to the last plate that is under the second dorsal spine and touching the ascending process. These correspond to plate positions 5 and 8 in previous nomenclature . All measurements were done with Vernier calipers accurate to 0.02 mm and had repeatabilities of 1.1% ± 0.9% (SD)(plate width) and 3.9% ± 2.9% (SD)(plate height). Plate width and height measurements on both sides of the body were summed and standardized by body length and depth, respectively. Similar QTL were detected when residuals from regressions of plate width and height on standard body length and depth were mapped. When raw plate width and height measurements were used, we also detected one additional significant QTL on LG 19 (plate width: LG 19, LOD = 5.42, 7.3 percent variance explained [PVE]; plate height: LG 19, LOD = 7.3, 11.6 PVE). Standard body length itself maps to LG 19 (LOD = 10, 13 PVE). The LG 19 effect on plate size is not significant when plate measurements are normalized by standard body length, suggesting that the LG 19 QTL is a general body size QTL, while the other size QTLs act on plate size separately from total body size. | 15069472_p19 | 15069472 | Morphological analysis and QTL mapping | 4.217577 | biomedical | Study | [
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All morphological traits in Cross 1 were analyzed with MapQTL 4.0 using the same parameters as described by Peichel et al. . Microsatellite markers that were closely linked to QTL detected in Cross 1 were genotyped in all Cross 2 animals . LOD scores between LG 4 markers and the major plate locus in Cross 2 were calculated using Map Manager v2.6.6 . Mean total plate numbers in low-plated fish that inherited different alleles at microsatellite loci on LGs 4, 7, 10, and 26 were compared using one-way ANOVA (Statview v5.0.1, SAS Institute Inc., Cary, North Carolina, United States). | 15069472_p20 | 15069472 | Morphological analysis and QTL mapping | 4.071686 | biomedical | Study | [
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The GenBank accession numbers for the Bmp6 gene is AY547294 and for the 12 additional new microsatellites Stn 210–219, 222–223 are BV102488–BV102499. | 15069472_p21 | 15069472 | Supporting Information | 2.3039 | biomedical | Other | [
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By charging authors a fee to have their work published in lieu of charging readers to access articles, open-access publishers such as the Public Library of Science (PLoS) and BioMed Central (BMC) have transformed the traditional publishing system. This reliance on a seemingly untested revenue stream has generated skepticism that authors will be both willing and able to pay publication charges. | 15094807_p0 | 15094807 | Publication Charges—Nothing New | 1.09165 | other | Other | [
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Publication fees are not a phenomenon born of the open-access movement. Many authors regularly pay several thousands of dollars in page charges, color charges, correction costs, reprint costs, and other fees to their publisher, even when such costs are entirely voluntary. In the EMBO Journal , for example, authors are allowed six pages of text free, but are then charged $200 per page beyond that. A review of recent issues shows that almost all authors exceed six pages, voluntarily paying on average over $800 to publish their articles. | 15094807_p1 | 15094807 | Publication Charges—Nothing New | 1.06236 | other | Other | [
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Furthermore, in addition to paying other publication charges, authors may be willing to pay extra for their articles to be made open access, as several publishers have recently recognized. A recent survey of authors in the Proceedings of National Academy of Science ( PNAS ) found that although PNAS already makes its content freely available after six months, nearly 50% of PNAS authors expressed a willingness to pay an “open-access surcharge” of $500 or more to make their papers available for free online immediately upon publication—this above and beyond the $1,700 in page charges that the average PNAS author already pays . | 15094807_p2 | 15094807 | Publication Charges—Nothing New | 1.12845 | other | Other | [
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Although we recognize that authors who submit to PLoS Biology may well be a self-selected group of enthusiastic open-access supporters, we have found that nearly 90% of those who submit manuscripts do not request a fee waiver, and the few who do still offer to pay some portion of the fee. | 15094807_p3 | 15094807 | Publication Charges—Nothing New | 1.060125 | other | Other | [
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The concern about authors' ability to pay publication charges will become less pressing as governments, funding organizations, and institutions increasingly support open-access publication on their researchers' behalf. More funding agencies are joining the Howard Hughes Medical Institute, the Wellcome Trust, and others who have already designated funds for open-access publication. (For more information about these funders' announcements and other international policy statements relevant to open access, see http://www.plos.org/openaccess .) | 15094807_p4 | 15094807 | Publication Charges—Nothing New | 1.146611 | other | Other | [
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Universities, too, are supporting open access directly by setting aside funds for open-access publication through institutional memberships with BMC and PLoS or through discretionary funds that faculty can tap into to pay publication charges. Such approaches reduce authors' reliance on individual grants to support charges directly and ensure equal access to publishing options that require such payments. | 15094807_p5 | 15094807 | Publication Charges—Nothing New | 1.12351 | other | Other | [
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] | en | 0.999999 |
Even with the steady increase in sources to pay publication fees, detractors claim that open-access publishing may lead to a situation in which some authors are simply unable to publish their work due to lack of funds. The response to this concern is that the ability of authors to pay publication charges must never be a consideration in the decision to publish their papers. To ensure that this happens, PLoS has a firewall in place such that neither the editors nor the reviewers know which authors have indicated whether or not they can pay. Because all work judged worthy of publication by peer review should be published, any open-access business model should be designed to account for fee waivers, just as publishers have always absorbed some authors' inability to pay page and color charges. PLoS grants full or partial publication-charge waivers to any author who requests them, no questions asked. | 15094807_p6 | 15094807 | The Disenfranchised | 1.120764 | other | Other | [
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] | en | 0.999996 |
In part, the savings to institutions, hospitals, nongovernmental organizations, and universities provided by open-access publications could help to establish funds for researchers who are less well supported. In the developing world, as free online access to scientific literature is increasingly seen as a political imperative, organizations such as the World Health Organization, the Oxford-based International Network for the Availability of Scientific Publications, and Brazil's SciELO are likely to become more willing to pay open-access publication charges for authors who cannot afford them. The Open Society Institute (OSI) already pays such costs for universities and other organizations in a number of countries in which the foundation is active by way of a PLoS Institutional Membership that grants waived publication charges to authors while providing compensatory revenue for PLoS. | 15094807_p7 | 15094807 | The Disenfranchised | 1.176269 | other | Other | [
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] | [
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] | en | 0.999998 |
Perhaps the real misconception about the unfair burden that open access places on authors resides in the terminology—the term “author charge” is itself misleading. Publication fees are not borne purely by authors, but are shared by the many organizations whose missions depend on the broadest possible dissemination and communication of scientific discoveries. Some of those may provide funding for open-access publication as intermediaries between authors and journals, as OSI does. Others—including many government-financed funding agencies—do so directly through their research grants to scientists. In both cases, funding open access is an effective way to fulfill mandates for public access to and accountability over scientific research and to ensure that all worthy research is published. | 15094807_p8 | 15094807 | The Disenfranchised | 1.295525 | other | Other | [
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While so far only one prion protein is known in mammals, several prion-like proteins capable of forming self-propagating aggregates have been found in various yeast species. The common structural feature of yeast prion proteins is the so-called prion domain, characterized by the high content of glutamines (Q) and asparagines (N) , also known as the Q/N-rich domain. The prion domains are the major structural determinants that are solely responsible for the polypeptide aggregation and propagation of the aggregates. Interestingly, the mammalian PrP Sc is fundamentally different from yeast prions, since it lacks a Q/N-rich domain, indicating that distinct structural features are responsible for its ability to form self-propagating aggregates. The Q/N-rich domains in yeast prions are transferable in that, when fused to a heterologous polypeptide, they confer prion properties to this polypeptide. With a low probability, soluble proteins with prion domains can change conformation to form self-propagating aggregates, which can be transmitted to daughter cells . As with PrP Sc , yeast prions efficiently recruit soluble molecules of the same species, thus inactivating them . Also with low probability, the aggregation-prone conformation of yeast prion proteins can reverse to a soluble functional conformation. Certain yeast prion proteins, when in soluble conformation, function in important pathways; e.g., Sup35 (forming [PSI + ] prion) controls termination of translation, and Ure2 (forming [URE3 + ] prion) controls some membrane transporter systems. Aggregation of these proteins leads to phenotypes (e.g., suppression of nonsense mutations or transport defects) inherited in a non-Mendelian fashion owing to the nonchromosomal basis of the inheritance. | 15094820_p0 | 15094820 | Prion Domains | 4.917628 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
A remarkable feature of yeast prion proteins is their ability to produce distinct inherited “variants” of the prion. For example, [PSI + ] prion could exist in several distinct forms that suppress termination of translation to different degrees. These “variants” of yeast prions are analogous to different prion “strains” of PrP Sc , which cause versions of the disease with different incubation periods and different patterns of brain pathology. The molecular nature of distinct PrP Sc strains is determined by specific stable conformations of PrP. Similarly, “variants” of yeast prions are explained by different stable conformation states of the corresponding prion proteins . Strict conformation requirements for aggregate formation can also explain interspecies transmission barriers, where prion domains of Sup35 derived from other yeast species cannot cause formation of [PSI + ] prion in Saccharomyces cerevisiae, in spite of a high degree of homology. This observation is very intriguing, especially in light of a recent finding that prion conformation of some proteins is required for formation of prions by the other proteins. For example, for de novo formation of [PSI + ] prion, a distinct prion [RNQ + ] should be present in a cell , probably in order to cross-seed Sup35 aggregates. This is in spite of relatively limited homology between the prion domains of these proteins. The apparent contradiction between the interspecies transmission barriers of very homologous prion proteins and possible cross-seeding of aggregates by prion proteins with more limited homology represents an interesting biological problem. On the other hand, this apparent contradiction may indicate that prion formation is a more complicated process than we currently think and that it may involve many cellular factors. | 15094820_p1 | 15094820 | Inheriting Variations | 4.781038 | biomedical | Study | [
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] | [
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Although yeast prions have been studied for almost ten years, very little is known about their biological significance. We do not know the functions of the majority of proteins that can exist as prions. Even if a function of prion proteins, such as with Sup35 or Ure2, is known, we do not understand the biological significance of their “prionization,” i.e., that they aggregate and propagate in the aggregated form. A very intriguing and unexpected finding was that formation of [PSI + ] prion causes a wide variety of phenotypic alterations, which depend on the strain background . In fact, comparison of yeast strains of different origin, each with and without [PSI + ] prion, showed that certain strains with [PSI + ] prion have different sensitivity to stresses and antibiotics than their non-prion derivatives, despite their genetic identity. In some strains, cells with [PSI + ] prion demonstrated better survival than their non-prion counterparts in the presence of inhibitors of translation or microtubules, heavy metals, low pH, and other deleterious conditions, which of course gives a strong advantage to the [PSI + ] cells. It is likely that some genomic mutations could be suppressed and therefore become silent when termination of translation by Sup35 is partially inactivated in [PSI + ] prion cells . [PSI + ] could also reveal previously silent mutations or their combinations. It was hypothesized that switches between prion and non-prion forms of Sup35 enhance survival in fluctuating environments and provide a novel instrument for evolution of new traits. | 15094820_p2 | 15094820 | What Do Prions Do? | 4.412509 | biomedical | Study | [
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Searching genomes of various species demonstrated that a relatively large fraction of proteins (between 0.1% and 2%) contain Q/N-rich domains or polyQ or polyN sequences. These domains are often found in transcription factors, protein kinases, and components of vesicular transport. The Q/N-rich domains usually are not evolutionary conserved and their functional role is largely unknown. Some of the Q/N-rich or polyQ domains facilitate aggregation of polypeptides, especially if expanded owing to mutations. Such expansion of the polyQ domains in certain neuronal proteins could cause neurodegenerative disorders, e.g., Huntington's disease or several forms of ataxia. Importantly, aggregates formed by polypeptides with the Q/N-rich or polyQ domains are not necessarily self-propagating aggregates, i.e., prions. In fact, there are additional structural properties of the polypeptides that provide the self-propagation (see below). Even if a protein with a polyQ domain does not form a prion, its aggregation may depend on certain prions. For example, recent experiments demonstrated that [RNQ + ] prion dramatically stimulated aggregation of fragments of recombinant human huntingtin or ataxin-3 with an expanded polyQ domain cloned in yeast . [RNQ + ] facilitated the nucleation phase of the huntingtin fragment aggregation, suggesting that this prion can be directly involved in seeding of the aggregates. The major question now is whether there are analogous prion-like proteins in mammalian cells that are involved in aggregation of huntingtin or ataxin-3 and subsequent neurodegenerative disease. | 15094820_p3 | 15094820 | Q/N Does Not Necessarily a Prion Make | 4.711321 | biomedical | Study | [
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The first indication that mammalian proteins with Q/N-rich domains can form self-propagating prions came from recent work with a regulator of translation cytoplasmic polyadenylation element-binding protein (CPEB) from Aplysia neurons . The neuronal form of this protein has a Q/N-rich domain similar to the prion domains of yeast prions. The Q/N-rich domain from CPEB (CPEBQ), when fused to green fluorescent protein (GFP), conferred upon it prion-like properties. The CPEBQ–GFP fusion polypeptide existed in yeast cells in one of the three distinct states, i.e., soluble, many small aggregates, or few large aggregates. Mother cells almost always gave rise to daughter cells in which the CPEBQ–GFP polypeptide was in the same state, indicating the ability of these aggregates to be inherited, i.e., to self-propagate. Furthermore, full-length Aplysia CPEB protein, when cloned in yeast, can also exist in two distinct states, soluble and aggregated, which is an inherited feature. Very unexpectedly, unlike other prions, the aggregated state of CPEB was more functionally active than the soluble form . These data strongly suggest that metazoan proteins with Q/N-rich domains are potentially capable of forming prions. The challenge now will be to establish whether CPEB can exist as a self-propagating aggregate in Aplysia or mammalian neurons. | 15094820_p4 | 15094820 | Q/N Does Not Necessarily a Prion Make | 4.49985 | biomedical | Study | [
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What makes protein aggregates in yeast propagate? The key cellular element that is critical for this process is molecular chaperone Hsp104 . This factor is specifically required for maintenance of all known prions within generations and probably is not involved in prion formation (i.e., initial protein aggregation). [PSI + ] yeast cells have about 60 seeds of this prion (although this number differed in different [PSI + ] isolates), and maintenance of about this number of seeds after cell divisions requires functional Hsp104 . In fact, in the absence of Hsp104, prion aggregates continue to grow without increase in number and are rapidly lost in generations . Since this chaperone can directly bind to protein aggregates and promote there disassembly , it was suggested that the main function of Hsp104 in prion inheritance is to disaggregate large prion aggregates to smaller elements, thus leading to formation of new seeds . Interestingly, although Hsp104 is conserved among bacteria, fungi, and plants, animal cells do not have this chaperone or its close homologs. Therefore, if yeast-type prions with Q/N-rich domains exist in animal cells, there should be alternative factors that disaggregate large prion aggregates into smaller species in order to keep the number of seeds relatively constant and thus maintain the prions. | 15094820_p5 | 15094820 | Mystery of Propagation | 4.57072 | biomedical | Study | [
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The fact that some proteins with Q/N-rich domains form self-propagating aggregates, while others can aggregate but cannot form prions, suggests that there should be some structural elements either within the Q/N-rich sequence or close to it that confer the ability to propagate. In an article in this issue of PLoS Biology by Osherovich et al. , the authors examined sequence requirements for prion formation and maintenance of two prion proteins, Sup35 and New1. They noted that both prion proteins contain an oligopeptide repeat QGGYQ in close proximity to Q/N-rich sequences and examined the functional significance of the repeats for aggregation and maintenance of the prions. In New1, in contrast to a deletion of the N-rich domain, deletion of the repeat did not affect aggregation of the protein or formation of the prion, but abrogated inheritance of the prion. With Sup35, the situation was somewhat more complicated, since repeats adjacent to Q/N-rich domain affected both protein aggregation and prion maintenance while more distant repeats affected only the prion inheritance. The authors suggested that the oligopeptide repeats facilitate the division of aggregates, either by serving as binding sites for Hsp104 or by altering the conformation of the polypeptides in aggregates to promote access for Hsp104 . | 15094820_p6 | 15094820 | Mystery of Propagation | 4.349273 | biomedical | Study | [
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The likely possibility was that the oligopeptide repeats could be interchangeable between different prions, leading to creation of novel chimeric prions. In fact, the authors constructed an F chimera, a fusion protein having the N-rich domain of New1 and the oligopeptide repeat of Sup35. This fusion polypeptide efficiently formed prion [F + ]. Furthermore, when the oligopeptide repeat sequence was added to a polyQ sequence, this fusion polypeptide also acquired the ability to form self-propagating aggregates. This work, therefore, clarifies the architecture of prions by defining two structural motifs in prion proteins that have distinct functions in aggregation and propagation. Interestingly, not all yeast prions have similar oligopeptide repeat motifs, indicating that distinct structures could confer prion properties to polypeptides that can aggregate. It would be important to identify these structures in order to understand the mechanisms of aggregate propagation. The work of Osherovich et al. may help to identify proteins from mammalian cells, plants, and bacteria that can potentially form prions. Finding these novel prions could be of very high significance since they may provide insight into a wide range of currently unexplained epigenetic phenomena. | 15094820_p7 | 15094820 | Mystery of Propagation | 4.545953 | biomedical | Study | [
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Advances in microarray technology have made the systematic study of expression levels of thousands of transcripts possible. This has been heralded as a major step forward in understanding the function of genomes, since transcript expression levels are expected to correlate with biological functions. Although this is clearly the case for many genes that change their expression in response to environmental stimuli , it is not known whether evolutionary changes in gene expression are determined primarily by Darwinian selection or by stochastic processes. Indeed, the extent to which natural selection has shaped the properties of organisms has been hotly debated ever since Charles Darwin proposed that organisms are adapted to their environment as a result of natural selection. At the molecular level, the view that most changes are due to Darwinian selection was challenged by Kimura's neutral theory of molecular evolution . This theory states that the vast majority of differences seen in nucleotide and amino acid sequences within and between species have no or only minor selective effects. Consequently, their occurrence within a species and the fixation of differences between species are primarily the result of stochastic processes. Thus, it is believed today that the evolution of the overwhelming majority of synonymous nucleotide changes within protein-coding exons, as well as changes in noncoding parts of genomes, are determined by mutational processes and random genetic drift . In fact, even at the level of morphology, it has been argued that many features are not adaptive, but instead result from physical constraints or historical accidents . However, since selection acts at the level of the phenotype while variation is generated at the level of the genotype, the proportion of changes caused by selection can be expected to be largest at the phenotypic level and smallest at the DNA sequence level. As a corollary, we may expect the proportion of selected changes to gradually decrease at the proteome and the transcriptome levels, since these are located progressively further from the phenotype. Consequently, a large proportion of transcriptome changes might be explained by historical accidents rather than by selective events. | 15138501_p0 | 15138501 | Introduction | 4.90516 | biomedical | Study | [
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] | en | 0.999996 |
To test whether this may be the case, we have investigated whether a neutral model can describe transcriptome differences observed among primate and mouse species as well as among various brain regions within a species. | 15138501_p1 | 15138501 | Introduction | 3.619008 | biomedical | Study | [
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If the majority of evolutionary changes are caused by historical accidents rather than by natural selection, they will accumulate mainly as a function of time rather than as a function of morphological or behavioral change of organisms. Applied to transcriptome evolution, a neutral model therefore implies that the rate of transcriptome change is proportional to time. In particular, if we assume that mutations cause changes in the relative amounts of transcripts independently of the absolute expression level of the gene, then the squared difference of the logarithm of the expression level is expected to increase linearly with divergence time . To investigate whether this is the case, we have studied differences in the gene expression levels of around 12,000 genes in the prefrontal cortex of six humans, three chimpanzees (Pan trogodytes), one orangutan (Pongo pygmaea), and one rhesus macaque (Macaca mulatta) using oligonucleotide microarrays. To exclude the influence of DNA sequence differences on the hybridization results, at least between humans and chimpanzees, only oligonucleotide probes that matched perfectly to the chimpanzee DNA sequences were used in the analysis ( see Materials and Methods ). In Figure 1 A, we plot species divergence times against the average squared difference between the logarithm of the expression levels of 1,998 genes that had expression levels large enough to be detected in all primate samples. Although comparisons involving orangutan and rhesus were complicated by nucleotide sequence differences to array probes, the result shows that the squared differences represent an approximately linear function of time over at least 20 million years. When we apply the same analysis to published gene expression data for the livers of three humans, three chimpanzees, and one orangutan , we again observe a linear relationship between gene expression differences and species divergence times . | 15138501_p2 | 15138501 | Transcriptome Evolution among Species | 4.277115 | biomedical | Study | [
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Since oligonucleotide-based microarrays are sensitive to DNA sequence differences and the orangutan and rhesus macaque genome sequences are not yet known—so that we cannot delete oligonucleotides carrying mismatches between the species—we used arrays containing around 28,000 cDNAs ranging in length from 500 to 1,500 nucleotides to assay gene expression patterns in the prefrontal cortex of six humans, five chimpanzees, five rhesus macaques, and five crab-eating macaques (Macaca fascicularis). Due to the greater probe length, these arrays are much less sensitive to DNA sequence differences and therefore can be used to compare gene expression in humans and macaques . When we plot the extent of gene expression divergence for 5,829 genes whose expression was detected in all samples against species divergence time, we again observe that expression differences accumulate approximately linearly with time . | 15138501_p3 | 15138501 | Transcriptome Evolution among Species | 4.134145 | biomedical | Study | [
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In a recent study of gene expression in the brains of humans, chimpanzees, and orangutans, we found that the rate of expression change on the human lineage has been larger than on the chimpanzee lineage . This is in apparent contradiction to the linearity observed here. However, the analysis of Enard et al. was based on less than 5% of all genes expressed in the brain because it was confined to genes that differed significantly in expression between humans and chimpanzees. In contrast, here we perform a transcriptome-wide analysis of all genes with detectable expression in several primate species. However, the slightly higher divergence of humans than chimpanzees from the two macaque species may reflect the previously reported higher rate of gene expression divergence on the human evolutionary lineage . However, additional experiments are necessary to exclude the possibility that this is caused by experimental artifacts. | 15138501_p4 | 15138501 | Transcriptome Evolution among Species | 4.148812 | biomedical | Study | [
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The clocklike accumulation of expression differences between species observed for primates is in agreement with the recent observation that differences in gene expression are consistent with phylogenetic relationships among Drosophila species , and both these observations are compatible with the predictions of the neutral model. However, under certain selection scenarios, positively selected changes would also accumulate linearly with time . Therefore, linear accumulation of expression differences alone does not rule out selection. | 15138501_p5 | 15138501 | Transcriptome Evolution among Species | 4.170428 | biomedical | Study | [
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In addition to the clocklike accumulation of evolutionary changes, the neutral theory states that the same forces determine the rate of evolution both within and between species . Thus, a neutral prediction with respect to transcriptome evolution is that genes that vary more within species should be more likely to change between species as well. In order to test this, we ranked 2,926 genes with detectable expression levels in six humans and three chimpanzees according to their variation within humans and calculated the species divergences for the 25% of genes that had the largest and the smallest human variation, respectively. Figure 1 C shows that the genes with high variation among humans changed significantly faster between species than the genes with low variation. The magnitude of observed expression differences may be influenced by DNA sequence mismatches affecting hybridization between orangutan and rhesus samples and array probes. However, the difference in divergence rates between genes with high and low expression variation within species is unlikely to be explained by hybridization artifacts, since this would require a difference in sequence divergence between the two groups of genes. | 15138501_p6 | 15138501 | Transcriptome Evolution among Species | 4.174745 | biomedical | Study | [
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We further considered the correlation between the average diversity within humans and chimpanzees and the divergence between the species for the 2,926 genes. This correlation is highly significant ( p < 0.001) as gauged by a permutation test ( see Materials and Methods ). Since all array probes that carried sequence differences between humans and chimpanzees were removed prior to analysis, this correlation is not affected by hybridization artifacts. The strength of the correlation (τ = 0.24) is of a similar magnitude as the one obtained for the correlation of diversity and divergence of random genomic DNA sequences in humans and chimpanzees (τ = 0.179, p = 0.028, n = 76), the vast majority of which are noncoding . Thus, although the two measures are not directly comparable, the degree of correlation between intraspecific diversity and interspecific divergence is similar for brain transcriptomes and random genomic DNA sequences in humans and chimpanzees. | 15138501_p7 | 15138501 | Transcriptome Evolution among Species | 4.134356 | biomedical | Study | [
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To investigate whether gene expression differences accumulate as a function of time also in another group of mammals, we analyzed three mouse species. An advantage in this case is that post mortem artifacts are less likely to influence the results than in the case of autopsy material of humans and great apes. We determined differences in gene expression levels for around 9,000 genes in the frontal cortex of six outbred Mus musculus, three outbred M. spretus, and one M. caroli. As shown in Figure 3 A, the squared transcriptome differences accumulated linearly with time among the mouse species. To test if divergence rates differ for the genes with high and low variation within species, we investigated the 25% of the 2,742 genes detected in all samples with the highest and the lowest variation within M. musculus, respectively, as was done in the primates. Figure 3 B shows that genes that vary more within M. musculus diverged faster among mouse species than genes that vary less. As in the case of primate species, imperfect matches of M. spretus and M. caroli mRNAs to the array oligonucleotides may partly influence the observed expression differences between species. Nonetheless, as for primates, the difference in divergence rates between genes with high and low expression variation within species is unlikely to be explained by hybridization differences since there is no indication that genes that vary more in expression within species diverge faster between species with respect to their DNA sequence. The correlation between diversity and divergence for M. musculus and M. spretus for genes detected in both species is highly significant (τ = 0.29, p < 0.001, n = 3,139), although in this case we cannot correct for DNA sequence differences. A correlation between gene expression differences within and between species was recently demonstrated also in teleost fish . Thus, in agreement with the neutral model, genes that vary more within species tend to vary more between species in three vertebrate groups. | 15138501_p8 | 15138501 | Transcriptome Evolution among Species | 4.330808 | biomedical | Study | [
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] | en | 0.999996 |
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